Methods in Cell Biology 2015

Johannes A. Schmid

Internet: www.meduniwien.ac.at/user/johannes.schmid

some contents contributed by:

Dr. Lukas Mach
Institut für Angewandte Genetik und Zellbiologie , 2
Univ. f. Bodenkultur

Overview of Topics

• scientific strategies and assay systems

• cell culture
• labelling and transfection of cells
• analysis of cellular components
• analysis of molecular interactions
• fluorescence measurements
• microscopy
• flow analysis (fluorescence activated cell sorting, FACS)
• analysis of various cellular processes (proliferation, apoptosis..)

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 Details of the lecture
1. Scientific strategies
2. cell culture
3. labelling and transfection of cells
a) radioactive and chemical labelling
b) transfections: overexpression of genes and gene suppression
c) reporter gene assays
4. gene-suppression (siRNA-technologies)
5. analysis of DNA and proteins (electrophoresis and blotting)
6. subcellular fractionation (centrifugations…)
7. methods to detect macromolecular interactions
a) Yeast 1- und 2-hybrid systems
b) co-immunoprecipitations
c) fluorescence resonance energy transfer (FRET)
8. methods of fluorescence measurements
9. realtime-PCR
10. transmitted light microscopy and contrast principles
11. fluorescence microscopy
12. confocal laser scanning microscopy
13. flow analysis (FACS)
14. analysis of various cellular processes (proliferation, apoptosis..)
15. methods to investigate vesicular transport processes
4

Nodes of regulation in cellular systems

ligands
receptors
cell membrane
oligomerization
signal transduction
transport
signal
transduction modified
protein
Golgi
transport posttranslational
activation
transcription modification
factor ER poly-ubiquitination
transport protein degradation

nucleus
translation
transcription splicing
pre-mRNA mRNA mRNA
transport
DNA
micro-RNA
processing
pre-micro-RNA micro-RNA 5

degradation

2
 General scientific strategies

• descriptive strategies: the whole system (cell, organism) is

observed without influencing it

advantage: physiological states are not altered

disadvantage: it is difficult to elucidate cause-effect relationships

• mechanistic (manipulating) strategies: various factors are kept

constant, while others are altered on purpose – the change in
the whole system is monitored.

advantage : cause-effect relationships can be monitored or

detected
disadvantage : the physiological steady state is altered and
influenced. Results might be artefacts of the measurement
system.

Experimental Systems I

• In vitro, biochemical systems: investigation in a solution

(e.g. enzyme reactions): many parameters can be fixed
(temperature, pH, buffer composition…).
Example: enzyme activity assay, EMSA …

• In vitro, cell biological systems: including cellular structures

e.g. in vitro-transcription/translation with membrane
components, in vitro-fusion of endosomes, nuclear import
assays with isolated nuclei etc. Experimental conditions
have to be set in a way that cellular structures are not
damaged (e.g. isotonic buffer, physiological pH….); reaction
partners can be influenced widely (e.g. antibodies can be
added…)

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 Experimental Systems II

• cell culture systems

- immortalized cell lines: unlimited passages

- primary cells: limited passage number, more

complicated cell culture (e.g. coated plates…)

- co-culture of different cell types:

e.g. keratinocytes + fibroblasts in a collagen matrix.

• xenograft systems:
- cells are injected into immuno-compromized mice
(nude mice, SCID mice); e.g. subcutaneously
- tissue recombination systems (e.g. prostate epithelial
cells with mesenchymal cells injected into the renal
capsule of SCID mice)

Experimental Systems III

• Organ cultures
(e.g. skin sheets,
brain slices…)

• Organ perfusions

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 Experimental Systems IV
• Animal Experiments:
Just the whole organism provides the full complex biological system that is
relevant for most biomedical research topics. The whole organism includes
superordinated systems such as the nervous system, the blood circulation,
the endocrine system and so on. The cells are in their normal organ
environment; cellular communications are intact…. – thus the highest
possible physiological state can be achieved. However, specific components
of the system (e.g. certain cells) are not easily accessible – and specific
experimental manipulations (e.g. of specific cells without side effects) are
often difficult). Cause-effect relationships are often difficult to elucidate –
and there is a big „black box“ due to the complexity of the system.
It has to be considered that results obtained with animals such as mice often
cannot be transferred to human beings despite the highly physiological
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system.

Experimental Systems V
• Special case: Transgene and knock-out animals :
Specific elimination of genes (knock-out) or incorporation of genes
(transgenes, knock-in) allow a better elucidation of cause-effect relationships.
However, classical knock-outs (eliminating a gene in all cells) – is often
embryonically lethal – and does not allow conclusions for its function in the
adult animal (e.g. mouse). Vice versa, it can happen that there is no
phenotype (if the function of the gene is taken over by another gene). Knock-
ins can be quite artificial as well (if the transgene is expressed at higher levels
with strong promoters). Modern approaches often aim for „conditional knock-
outs“ or knock-ins: In most cases the Cre-recombinase / loxP system is used
for that purpose:
Conditional knock-out: the gene (or a crucial exon) is placed between loxP
sites – Cre recombinase (which can be expressed in specific organs or cells by
organ specific promoters) cuts out the gene – thus the gene is deleted just in
a certain organ or cell type; using inducible Cre, this system allows gene
deletion at a defined time point (e.g. when the animal is adult).
Conditional knock-in: the gene is placed behind a Stop-cassette, which is
flanked by loxP sites. Without Cre activity, the gene is not expressed, with Cre
activity (organ specific), the Stop cassette is excised and the gene is expressed
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 Conditional transgene mouse models with the
Cre / loxP system
1. Cre mouse strain
2. loxP-mouse strain
• Cre recombinase cuts out
• Endogenous genes can be
sequences between loxP sites
flanked by loxP sites (using
(or inverts sequences between
recombination techniques –
inverted loxP sites)
e.g. affecting essential exons)
• Cre expression can be > conditional knock-out
rendered cell-type or organ-
• Genes can be overexpressed
specific using cell-type specific
conditionally by inserting an
promoters driving Cre
expression construct headed
expression (spatial control)
by a loxP-flanked „Stop
• Cre expression can be made cassette“, which is cut out by
inducible by using chimeras of Cre recombinase
Cre with mutated estrogen
receptor domains
(temporal control:
e.g. Cre-ERT2) 12

Examples for conditional mouse models

cell-type specific
promoter
mER Cre mER Cre-ERT2

mER: mutated estrogen receptor (responds to tamoxifen as ligand; without

the ligand it keeps the Cre in the cytosol > inactive; upon addition of
tamoxifen, the nuclear localization sequences of mER become active leading
to translocation of the chimera into the nucleus, where Cre recombinase can
exert its function on loxP-flanked DNA sequences).

conditional knock-out conditional transgene

Gene of interest Stop Gene of interest

loxP loxP good promoter

(e.g. pCAGGS)

mER Cre mER mER Cre mER

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Tam Tam Tam Tam

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 Specific cell ablation or cell labeling
in transgenic mice

loxP loxP

Stop DTR (Diphteria toxin receptor)

good promoter
(e.g. pCAGGS)

cross-breeding with a cell-type specific Cre

strain
DTR is expressed only in specific cell types
injection of diphteria toxin leads to specific
killing of these cells
loxP loxP

Stop EYFP (enhanced yellow fluorescent protein)

good promoter > fluorescent labeling of a specific cell type of interest

(e.g. pCAGGS)
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Genome Editing for generating transgene

animals

• Novel methods to edit genes directly in the genome, e.g. using

CRISPR/Cas9 technology or Zn-finger nucleases, or TALENs
allow faster and even multiplexing type manipulations (e.g.
targeting 5 genes simultaneously)
• Genome editing is then usually done in ES-cells, which are
subsequently injected into blastocysts.

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 Cell Culture Methods

• cell culture of immortalized cells

• cell culture of primary cells (often just a few passages)
• culture of polarized cells
• co-culture of different cell types

primary fibroblasts transformed

(from skin dermis) fibroblasts 16

What you need for cell culture

• Incubator (37°C) with CO2-

supply and humidification

• Sterile bench (Laminar Flow: a

laminar flow of filtered air keeps
the bench sterile) – has to be
switched on approx. 10 min
before using it, the filter has to
be checked from time to time
(Particle Measurement)

• inverted cell culture microscope

(4x, 10x, 20x objectives)

• L2-biosafety (e.g. for virus-

work): not only incoming air is
filtered, but also the air that
leaves the laminar flow. 17

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 Culture Media

mammalian cells need media with vitamins, amino

acids, hormones and growth factors
Serum, mostly fetal calf serum (FCS), is the source of
growth factors (such as FGF, EGF…)
Common composition
- DMEM (Dulbecco‘s Modified Essential Medium)
- 10% FCS
- 2 mM Glutamine (unstable amino acid, has to be added
again, if the medium is older than app. 6 weeks)
- Penicillin (100 u/ml)
- Streptomycin (100 µg/ml)

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Fetal Calf Serum (FCS) Special Growth

has high concentrations of growth factors Factors
(e.g.: EGF, epidermal growth factor; FGF,
fibroblast growth factor; IGF, insulin-like Nerve- Growth Factor
growth factor) (NGF):
low amount of antibodies → compatible for neuronal cells
with cells of other species Hepatocyte-Growth
complement system is inactivated by a Factor (HGF): induces cell
heat shock (30 min., 56 °C) division of hepatocytes

disadvantage of FCS: expensive (≈ 100 € Keratinocyte-Growth

per Liter) Factor (KGF): for culture
of skin epithelial cells
might contain contaminants such as (keratinocytes)
tetracyclin (important if you use a Tet-
inducible cell culture system > purchase
guaranteed Tet-free FCS)
Alternative sources of serum: normal calf
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serum, horse serum…

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 Adhesion factors

adherent cells versus suspension cells

cell culture dishes: usually made hydrophilic
(charged groups) – sufficient for most cell types
collagen; Gelatine (denatured collagen)
sometimes necessary for good attachment (e.g. for
primary endothelial cells)
Components of extracellular matrix:
Fibronectin, Laminin – often better than collagen.
special case: „Feeder“-cells (irradiated) – or co-
culture of cells (e.g. fibroblasts in a collagen matrix
with keratinocytes on top)
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Contaminations in cell culture

Bacteria: might be a problem, when
they are resistant against the antibiotics
that are used (mostly Pen/Strep) >
then you have to use other antibiotics
(e.g. kanamycin)
Mycoplasm: procaryotes of an ancient
evolutionary stage, which do not have a
normal bacterial cell wall (therefore
they are resistant against Penicillin)
(can be eliminated with Kanamycin)

Yeast: rare (might occur when yeast

and mammalian cell culture are not
strictly separated)
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Fungi: quite rare

10
 Unnoticed mycoplasm contaminations can screw up experimental results

> Tests for Mycoplasm-Contamination

DNA-staining with DAPI or Hoechst 33258

no Mycoplasm
Mycoplasm

Alternative:
PCR- detection of Mycoplasm-DNA
(commercial kits are available) 22

Cell culture of polarized cells

the cells have to build the

tight junctions for building up
apical side the polarity.
tight junction Often they are cultivated on
lateral side filters, where the two sides
are accessible.

basal membrane

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 Methods to investigate polarized cells

Model systems
1. simple cell culture: just one side of the cell is accessible.
2. cell culture on microporous membranes: both sides are
experimentally accessible, the establishment of a tight
polarized monolayer can be checked be measuring the
electrical resistance between the two sides.
3. Organ cultures: e.g. Living Skin Equivalent
4. Organ perfusion: e.g. perfusion of isolated rat liver

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Culture of polarized cells on membranes

Measurement of electrical
resistance

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Cultivation of polarized cells on electrode
chambers

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Cultivation of polarized cells on electrode

chambers

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 Organotypic culture: Example: Skin Equivalent
Keratinocytes (cells of the upper layer of skin, the epidermis) are
seeded onto a collagen matrix, which contains fibroblasts (dermis
cells). As soon as they build a monolayer, they are elevated to the
surface of the medium (with their upper side exposed to air) – this
induces cell differentiation and the formation of a pseudo-epidermis
with several cell layers.

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Organ Perfusion
Perfusionsdruck (cm H 2(cm
perfusion pressure O) H2O)
Liver
LEBER
Pump
Peristaltikpumpe
bile canula
Gallengangskanüle Thermosensor
Polarized cells such as hepatocytes
V. porta
V. porta can be maintained in their organ
Kanüle
architecture maintaining their
V.V.cava
cavaKanüle
canula
polarity. The organ is perfused
using glas capillaries linked to the
normal blood vessels the supply
Computer Fraktionskollektor
fraction collector
the organ with blood (and
nutrients/oxygen). A buffer (37°C,
gas humidification
Gas-Befeuchtung
temperature percolated with O2) containing the
Temperatur-
recording
Messung nutrients can be perfused through
the liver. Marker substances can
be applied and their transport
from the blood side (basolateral)
to the bile side (apical) can be
determined.
Schreiber

Water Bath
Wasserbad
Perfusion Buffer
Perfusionspuffer 93% O 2
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7% CO 2

14
 Example of an experiment with polarized cells

Transmigration of Leukocytes through a Monolayer of polarized

endothelial cells
Leukocytes are labeled with a
fluorescent marker (e.g. CFSE
– an ester, which is turned
fluorescent just after uptake
into cells due to esterases)

Cells are seed onto a layer of endothelial cells (e.g. after activating the
endothelial cells with inflammatory cytokines – which leads to the
synthesis of adhesion molecules on the surface of the endothelial
cells). Adhesion of leukocytes leads to transmigration into the lower
chamber. The extent of the transmigration can be determined by lysing
the cells and quantifying the fluorescence (or by counting the
fluorescent cells) 30

Methods for labeling of cellular components

• Radioactive labeling: e.g. with radioactive amino acids (35S-

Methionin…), which are incorporated into newly synthesized
proteins. Pulse/chase experiments can give insights into half life,
transport. Processing… of proteins.
• Internalisation of high molecular weight markers (loading of
endosomes and lysosomes).
• Labeling of cell surface proteins (e.g. by biotinylation with cell-
impermeable reactive biotin compounds).
• Addition of cell-permeable labeled substances, which integrate into
specific structures (e.g. Golgi-specific fluorescent lipids)
• Transfection of cells with expression plasmids
• Protein-Transduction (cell-permeable peptides)
• Micro-Injection of substances.

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 Radioactive Labeling of Biomolecules

Amino acids: [35S]Methionine, [3H]Leucin

Monosaccharides: [3H]Mannose, [3H]Glucosamine

Phosphate: [32P] phosphoric acid

Alternative: 33P: lower radioactivity

Sulfate: [35S]Sulfuric acid

Fatty acids: [3H]Palmitinic acid

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Radionuclides

33

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 Metabolic labeling with amino acids

usually [35S]Methionine labeling

advantage: high specific radioactivity
disadvantage: relatively rare amino acid > check first,
how many methionine are in the protein! (e.g.
Ubiquitin: only 1 Methionine, but 9 Leucines)
requires Methionine-free culture medium for depletion
of endogenous stores by pre-incubation
(addition of dialyzed FCS)

34

Metabolic Labeling with Monosaccharides

[3H]Mannose or [3H]Glucosamine

Activated in the cytosol by coupling to nucleotides →

GDP-Mannose, UDP-N-Acetyl-Glucosamine (UDP-
GlcNAc)

requires Glucose-poor culture medium

Addition of alternative energy sources

(Glutamine, Pyruvate)
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 Pulse-Chase-Experiments

preincubation without marker (depletion of

endogenous stores)
Addition of the labeled substance → "Pulse" (5 min -
1 h or longer)
Stop of the "Pulse" by addition of an excess of
unlabeled compound
further incubation → "Chase" (min - 48 h)
Stop of culture → Analyses

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Example for a Pulse-Chase-Experiment

Fibroblasts of patients, were labeled with 35S-methionine:

Pulse: 1 h at 37°C
Chase: 7 h at 19°C and 37°C (19°C inhibits transport from trans-Golgi to
late endosomes/pre-lysosomes) > Immunoprec. and autoradiography

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 Iodination of Proteins (Labeling with 125J)

direct: modification of Tyrosine with [125J]:

- chemical oxidation: Chloramine-T oxidizes Na125J
and leads to iodination of Tyrosine
- enzymatic: Lactoperoxidase: oxidizes Na125J in
presence of H2O2.
indirect: Modification of amino groups (Lysine, N-
Terminus) with [125J]-labeled Bolton-Hunter-Reagenz
(N-succinimidyl 3-(4-hydroxy 5-[125J]iodophenyl)-
propionate)

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Biotinylation

High affinity binding partner of Avidin and

Streptavidin (> easy purification and
detection by beads, coated ELISA-Plates…)
Streptavidin has 4 binding sites for biotin
(complexes can be formed with bivalent
biotin-linkers!) > signal amplification is
possible (ABC: avidin-biotin-complexes)
Most commonly used method to label cell
surface proteins
Biotinylation of amino groups with Biotin-
Hydroxysuccinimid-Esters or of cysteines
(SH-group) by Maleimide-derivates

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 Labeling of Endosomes, Lysosomes

• By high molecular weight compounds, which are easily

detectable, not permeable for the cytoplasmic membrane and
taken up by endocytosis
Examples:
a) unspecific internalization:
- FITC-Dextran (Fluorescein-labeled)
- Enzymes (Peroxidase…)
b) Specifically by receptors

Pulse/Chase conditions can be used to labeled specifically early

or late endosomes or lysosomes (e.g. using temperature
blocks....)

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Transfections
Usually designates the incorporation of DNA into mammalian cells. DNA present
in form of plasmids.
Transient Transfection: plasmid remains outside of the genome and is slowly
lost (degradation, dilution by cell division), exception: episomal replication –
e.g. SV40-Plasmids in COS-cells). The transfection efficiency varies – but can
reach close to 100%
Stable Transfection: integration of foreign DNA into the genome (Efficiency:
usually below 0.1%). Isolation of stably transfected clones requires selection
genes (for antibiotic resistance, e.g. puromycin, G418…). Plasmids are usually
linearized before transfection to increase the possibility of correct integration.

41

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 Example for a mammalian expression plasmid
(Replication origins no shown)

CMV-Promoter
poly-adenylation signal reporter gene

selction gene Multiple

(bacterial Cloning Site
selection with
Kanamycin,
mammalian-
selection with target gene
G418)

SV40-Promoter

suited for linearization poly-adenylation signal

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Selection markers for stable transfections

•Aminoglycoside-Phosphotransferase: Resistance against Neomycin

(bacteria) und G418 (mammalian cells). Selektion with G418 takes
quite long (app 2 weeks). Surviving colonies are isolated and further
cultures under selection pressure.
•Hygromycin-Phosphotransferase
•Puromycin-Acetyltransferase
•Dihydrofolat-Reductase (DHFR): Selection with Methotrexat; allows
amplifications of the target gene.

•GFP-Fusion proteins: fluorescence

can be used for selection: example:
EGFP-NF-κB expressing CHO-cells

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 Chemical Transfection Methods
Ca2+
Ca2+
• DNA-Calcium precipitates: at exact pH and
Ca2+-concentration: High efficiencies with
293-cells (90% and more), expression levels
are usually moderate. Ca2+

• Liposome mediated transfection: kationic

lipids which bind the negatively charged DNA,
and which are taken up as liposomes
Quite high transfection efficiencies but also
sometimes toxic effects and potentially
artefacts by high expression levels or
alteration of cellular membranes. (e.g.
Lipofectamine, Fugene...)

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Buffers
• HeBS-Buffer (Hepes buffered saline)
8 g NaCl - 280 mM final concentration
Protocoll: Calcium-Transfection
0.2 g Na2HPO4.7H2O (or 0.107 g anhydrous) - 1.5 mM
6.5 g Hepes (Sigma H-7006) (or 5.96 g of free acid) - 50 mM
400 ml A.dest.
Adjust the pH to exactly 7.05 (calibrate pH-meter with pH 4.01 and pH 7.00 buffers before). Add A.dest. to 500 ml,
filter through 0.2 µm filters and store in aliquots at -20°C (not longer than 6 months). Thawed aliquots shouldn't be
frozen again.
• CaCl2: 29.4 g CaCl2.2H2O (MW=147) in 100 ml A.dest (final conc.: 2 M) Filter through 0.2 µm filters and store
aliquoted at -20°C.
• Chloroquine (optional): chloroquine. 2H2O (Sigma C-6628): 12.9 mg/ml in PBS (conc.: 25 mM). Filter through
0.2 µm filters and store at -20°C.

Procedure (amounts are given for 6-wells):

1. Seed cells (about 500 000 cells per 6-well = per 10 cm2 ) one day before the transfection (in DMEM/10% FCS)
2. (Optional: 1 h before transfection, exchange the medium for medium containing 25µM chloroquine)
3. Thaw HeBS and CaCl2 at room temperature
4. For each transfection prepare aliquots of 71 µl HeBS
5. Prepare the DNA/ CaCl2-Mix: 4 µg DNA (total) in 62 µl A.dest. + 9 µl CaCl2
6. Add the DNA/ CaCl2-Mix drop-wise to the HeBS aliquots (by screwing the Gilson pipette) and slightly mix after
each drop. Incubate for 2 - 3 min at R.T. to form the DNA-precipitate (not longer).
7. Add the DNA-precipitate drop-wise to the cells (by screwing the Gilson pipette and moving it to cover the whole
surface of the cell culture; don't swirl the dish).
8. Carefully transfer the dish back to the incubator. Incubate for 24 h (or in the presence of chloroquine: for 10 h)
and exchange the medium afterwards. (The transfection is in the presence of FCS!). The efficiency of transfection is
in the range of 70-90% for 293 cells. Harvest the cells after 48 h. The protocol is adapted from Neil Perkins who
adapted it from Gary Nolan in 1995
(See web site: http://www.stanford.edu/group/nolan/ or CP in Mol.Biol. 9.1 and 9.11.2-3)
45

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 Lipofectamine2000 - Standard conditions

Transfection in presence of serum (e.g. 24h – 48h)

46

Chemical Transfection Methodes II

• Dendrimeres: charged Polymeres that bind

DNA (e.g. Superfect / Qiagen)

• complexes with DEAE-Dextran (polycationic

Dextran)

• poly-ethylene-imine (PEI):

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23
 Physical Transfection Methods

• Electroporation: common with

suspension cells, electro-
requires much DNA cuvette

Special case: Amaxa-

Nucleofection (now Lonza)

• Particle-gun (“Gene Gun”):

DNA on gold particles shot by
pressure onto cells (e.g.
neurons in brain slices).

• Micro-Injection of DNA: into

single cells (just limited cell
number can be targeted).
48

Viral Transfection Methods

• Adenoviruses, Retroviruses,
Lentiviruses:
Viruses developed fancy mechanisms to
get into cells – these are applied for
gene transfer. Virus-constructs are
generated, which contain the target gene
but not genes for virus replication (genes
essential for generating the viral particles
are supplied by “Packaging Cells”).

• Adenoviruses: transient Expression

• Retroviruses: stable integration but
target just proliferating cells
• Lentiviruses: stable integration, also
transduce quiescent, non-proliferating
cells.
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 Adenovirus Retrovirus

•Episomal gene expression •Long-term, stable gene expression; inheritable

•Infects dividing & •Infects dividing cells only

nondividing cells

•High-level protein •Moderate protein expression

expression

•Viral titers of up to 1012 •Viral titers of up to 106 cfu/ml (Can be

pfu/ml concentrated to 109 cfu/ml)

•Accommodates inserts of up •Accommodates inserts of up to 6.5 kb

to 8 kb

•Elicits immune reactions in •Does not elicit immune reactions in vivo

vivo
50

Example of and
adenoviral system

51

25
 Adeno-associated virus (AAV)

• is a small virus which infects human cells

• is not causing any obvious disease
• causes a very mild immune response
• can infect both dividing and quiescent cells
• AAV vectors persists mostly in an extrachromosomal state
without integrating into the genome of the host cell (the native
virus can integrate to some extent into the host genome).
• Promising gene therapy vectors (clinical trials have been done
for CFTR, hemophilia B, arthritis …)

52

53

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 Protocol:
Production of retrovirus by transfection of packaging cells

Packaging cell line: Phoenix cells. These cells must not be too confluent since this will lower the
production of virus significantly.
Day 1
Plate phoenix cells on 15 cm plate (1.5-1.75 x 107 cells)
Day 2
((Transfect phoenix cells using 25 µg DNA and 75 µl Fugene6 + 1.8 ml OptiMEM.
Incubate 30 min RT and add to cells.)) OR better use CaPO4!!!
Day 3
Carefully remove old medium and add 25 ml of fresh medium.
Incubate at 32°C for 24h or 48h (or 72h).
Day 4-5
Harvest supernatant after 24-48h. Virus sup can be harvested until the cells start looking unhealthy.
Put supernatant in 50 ml tube in ice bucket in the hood. Add 22 ml fresh medium to the packaging
cells and put back into incubator.
Spin viral sup to pellet any remaining cells.
For storage of virus: transfer to cryotube and snap freeze with N2 store at -80°C. Upon freezing virus
titer goes down roughly twofold!
For use straight away: Filter virus sup through a 0.45-µm cellulose acetate or polysulfonic filter
(do NOT use nitrocellulose filter since it binds proteins in the retroviral membrane!). Keep on
ice until use.
54

Protocol II:
Infection of MEFs with recombinant retrovirus for
making stable cell lines
Day 3
Split the target cells into 10 cm dishes.
Day 4
Add 8 µg/ml polybrene to the filtered viral supernatant. Mix gently by
inversion.
Replace the media in the target cells with the viral sup + polybrene.
Incubate at 37°C for 6h and add equal amount of media containing
polybrene.
Day 5
Replace the media.
Day 6
Replace the media with fresh media containing pyromycin to select for
transfectants. The amount of pyromycin used is determined by killing curve
experiments. Select colonies of stable transfectants keeping the pyromycin
in the media. 55

27
 Reporter-Gene Assays
Enzymes or other molecules, which are easily detectable, are
applied as „reporter molecules“ to detect promoter activities or the
activities of signaling pathways.
The reporter gene is cloned into an appropriate plasmid (e.g.
mammalian expression vector) and transfected into the cells of
interest, followed by the biological experiment (e.g. stimulation).
Examples for reporter-genes:

• EGFP (Enhanced Green Fluorescent Protein) and variants thereof (e.g.

destabilized EGFP)
• Luciferase
• CAT (Chloramphenicol-Acetyl-Transferase)
• ß-Galactosidase (lacZ)
• SEAP (secreted alkaline phosphatase) etc.

56

Regulated Promoters in Reporter Gene Assays

(artificial promoters)
• Tandem Repeats of transcription factor binding sites (e.g. 5x NF-kappa
B). Often commercially available (e.g. from Stratagene or Clontech)
> can be used to determine the activation of a certain transcription
factor (or signaling pathway)

minimal
promoter Reporter Gene
element

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 Regulated, Natural Promoters in Reporter Gene
Assays
• Natural promoters usually contain binding sites for several
different transcription factors (sometimes several copies of a
single TF binding site) > are regulated by several signaling
pathways.
Example:

NFκB
IRF-1 GRE AP-1 NF/IL-6
IL-8
Promoter Luciferase

p65 / c-Rel

> can be used to determine the regulation of a specific promoter of interest

Luciferase is usually easier to measure with high sensitivity than the gene of
interest (in this case IL-8)
58

Normalization constructs with constitutive promoters

…usually used for the normalization control in reporter gene assays

(to compensate for differences in transfection efficiency, extraction efficiency
or viability of cells in different samples)

Usual setting: Pathway-specific reporter construct (e.g. with firefly luciferase)

+ constitutive normalization construct (e.g. Ubiquitin-Promoter driven β-
galactosidase or Renilla-luciferase) > quantification of normalized values
(Luciferase / β-galactosidase or Firefly / Renilla-luciferase)

Promoters for the normalization vector:

• CMV: from human Cytomegalo-Virus: induces strong constitutive
expression (fast, about 1 day after transfection)
• RSV: from Rous Sarkoma-Virus: weaker, but very constant, constitutive
expression, slightly slower (takes 2 d)
• SV40: Simian Virus 40-Promoter
• Actin-Promoter: human promoter of a „housekeeping gene“
• Ubiquitin-Promoter: human promoter of a „housekeeping gene“
59

29
 Example of an Reporter Gene Assay

50 46.36
45 EP-cells
DU145
Luciferase/β-Gal
40

fold of control vector

35
30 26.66
25
20
15
10
4.63
5 1.94 0.90 0.49
0
p53 Rb E2F

Activities of different signaling pathways or molecules (p53, Rb, E2F) are

assessed with reporter constructs containing respective transcription
factor binding sites upstream of a luciferase vector.
For normalization purposes a constitutively expressed control gene has
to be cotransfected (e.g. β-galactosidase downstream of a constitutive
promoter e.g. ubiquitin-promoter).
Values are calculated as Luciferase/β-Gal. 60

Reporter-Gene Assay Systems for Analyses of

Signaling Pathways
Transcription factor construct
Gene of interest Transcription factor construct
Reporter-
Plasmid

In case that the gene of interests

activates the specific pathway,
which leads to phosphorylation
Reporter-
and activation of the used
Plasmid
transcriptionfactor construct, the
expression of the reporter (e.g.
luciferase) is induced.
61

30
 Detection of reporter genes: GFP (and variants)

• Fluorescence measurement by
fluorometry (e.g. in 96-well
fluorescence readers)

• Microscopy

• Flow analysis (cytometry,

FACS: Fluorescence activated
cell sorting: GFP-containing
cells can be separated from
other cells and if necessary
also further cultivated after
purification)
GFP fluorescence >
62

Detection of Luciferase (Firefly or Renilla Luciferase)

• Very sensitive detection in cell extracts by measuring the

luminescence generated from luciferase in presence of
Luciferin luciferin and ATP. The substrate Luciferin has to be injected
into the sample, and measured immediately (e.g. by
integrating for 5 sec) as the emitted luminescence decays
quickly. Emitted photons are measured with photomultiplier
Luciferase- tubes.
ATP PMT
Measuring devices: Luminometer
Zellextrakt (also as 96well devices available)

63
PMT: from http://www.molecularexpressions.com

31
 Detection of β-Galactosidase (lacZ)
• often by photometry (e.g. in ELISA
readers) using a yellow substrates, which
turns red in presence of Galactosidase
(substrate: CPRG, Chlorophenolred-β-D-
Galactopyranoside). Detection at 595 nm.
• Alternative: Chemiluminescence
measurement
• Alternative: Fluorimetric Detection:
Substrate: e.g. 3-carboxyumbelliferyl
beta-D-galactopyranoside, CUG is
converted to a fluorescent product
• Microscopical detection: with X-Gal (5-
Bromo-4-chloro-3-indolyl-ß-D-galactoside)
as substrate: produces a dark blue
reaction product
64

β-Galactosidase Assay with CPRG

Lyse cells
(recommended cell lysis buffer: 0.25M Tris/HCl pH 8.0, 0.25% (v/v) NP40,
2.5 mM EDTA)
Pipet about 10 µl of extract into a 96-well plate (appropriate neg. control / blank: 10 µl of mock
transfected cells, or non-transfected cells – as there is a slight endogenous b-Gal activity) – leave one
well empty for blank (A-1)
Add 100 µl substrate solution to the wells (also to the blank-well)
Incubate until red color develops (min to hours – depending on b-Gal activity, if you have low activity
you can also incubate at 37°C)
Optional: Stop with 50 µl of Stop solution (only necessary if you want to time it exactly, e.g. by adding
the substrate in a timed way and stopping the reaction in the same way)
Measure with ELISA Reader at 570 nm (Filter #3) optimum: 595 nm
Lysis Buffer:
0.25M Tris/HCl pH 7.4 (or better 8.0)
0.25% (v/v) NP40
2.5 mM EDTA
CPRG-substrate solution: 1 mg/ml (= 1.65 mM)
in PBS + 10 mM KCl, + 1 mM MgCl2
alternative substrate buffer:
60 mM Na2HPO4 pH 8.0, 1 mM MgCl2, 10 mM KCl, 50 mM Mercapto-ethanol
65
Stop solution: 0.5M Na2CO3

32
 Methods to suppress gene expression
(RNA interference)
Antisense-Technologies: to suppress gene expression were started many years
ago. Early approaches used antisense-oligonucleotides – but the effect was very
variable.
Alternative approaches used long antisense-strands hybridizing to the mRNA:
This usually leads to downregulation of gene expression – but quite often not
only for the targeted gene – but also unspecifically for other genes. The reason
is that this is „sensed“ by the cells like a long viral dsRNA, leading to virus
defense mechanisms: activation of PKR (protein kinase R), phosphorylation of
translation factors and general downregulation of protein synthesis.

Some years ago, scientists found that small dsRNA in the range of 19-21
nucleotides interferes specifically with target genes, without affecting other
genes (small interferent RNA, siRNA) – because they are too small to activate
virus defense mechanisms.

66

Principle of RNA-interference

Small dsRNA (siRNA) binds to an

RNA-induced silencing complex
(RISC); consisting of argonaute
proteins.
Together with the RISC, one RNA
strand binds to the target mRNA
and leads to specific degradation
of this mRNA.
The RISC complex is „recycled“
leading to degradation of addition
mRNA-targets.

67

33
 micro-RNA‘s – the biological mechanism to
suppress gene expression

miRNAs – can have

two effects:
1) mRNA degradation
2) inhibition of
translation (this
effect often does
not require a
100% match with
the target mRNA!)

Chemically synthesized siRNA

Design: traditionally the first AA-Duplett is searched – and the following 19
bases are checked for GC-content (should be 40 – 50%), the sequence
should be target gene specific (checked by BLAST) – an appropriate RNA
sequence and the reverse complementary RNA are chemically synthesized
(and ordered by a company, e.g. Dharmacon, Invitrogen, MWG,…).
Company homepages often offer a basic siRNA design:

http://www.dharmacon.com/
http://www.mwgbiotech.com/html/s_synthetic_acids/s_rna.shtml

The two short RNAs are annealed and transfected (usually using
methods that are suited for short oligonucleotides (e.g.
Lipofectamine2000 from Invitrogen, XtremeGene from Roche…).

69

34
 Vector-coded siRNA > small hairpin RNA (shRNA)

•the normal transfection methods, optimized for plasmids can be used

•Antibiotics selection genes (e.g. G418, puromycin…) can be included > stable
„knock-down“ cell lines can be generated (www.imgenex.com,
http://www.oligoengine.com/)

70

small hairpin RNA: hairpin-loop

71

35
 Professional Design of siRNA or shRNA
• Design via company website
http://www.thermoscientificbio.com/design-center/?redirect=true
This delivers a list of several possible sequences (gene specific,
checked by BLAST) – with a score based on empirically determined
criteria: Nature Biotechnology 22, 326-330, 2004
• Check literature for functional siRNA sequences
• For transduction of primary cells: lentiviral shRNA constructs
(also work in non dividing cells)
• there are also inducible lentiviral constructs available
(http://tronolab.epfl.ch/)
• Many vectors can also be obtained from plasmid repositories:
Addgene: http://www.addgene.org
Belgian repository: http://bccm.belspo.be/db/lmbp_search_form.php

72

Gene replacement strategy

5‘UTR 3‘UTR
gene of interest endogenous mRNA

siRNA
targeting the endogenous
mRNA via the
untranslated region
Expression plasmid containing:

good
foreign
promoter
3‘UTR the mutated gene
mutated gene of interest replaces the endogenous
(SV40 gene
PolyA)

73

36
 Important controls in siRNA experiments

• scrambled siRNA as negative control

• mutated siRNA with some mismatch as negative control (note:
might act as miRNA !)
• other siRNAs targeting the same mRNA should have the same
effect
• if you use shRNA (vector based RNA-interference) use an
unrelated shRNA as negative control (e.g. shRNA vector with
scrambled siRNA sequence).
The empty shRNA vector is not a valid negative control

Alternative Methods to influence endogenous

protein levels

Micro-Injection:
This allows injecting antibodies
against certain endogenous
proteins > interfering with their
functions. However, just a limited
number of cells (e.g. up to
hundreds with automated systems)
can be targeted > the following
analysis should be a single cell-
based assay, such as microscopy.

75

37
 Research Methods - Overview

• cell culture systems

• labelling and transfection of cells
• analyses of cellular components
• analyses of molecular interactions
• fluorescence measurements
• microscopy
• flow analysis (FACS)
• analyses of cellular processes (proliferation, apoptosis..)

76

Analysis of Proteins by SDS-PAGE

S-S

reducing agent (DTT) SO4-

or Mercapto-Ethanol
- breaks disulfide SDS – sodium dodecyl
bonds sulfate: coats proteins
95°C with negative charges

- - - -
- - - - -
-
- - - - - -
- -

77

38
 SDS-Gels
For final concentration of gel ( % T):

Stack gel
Separating gel (10 ml) (10 ml)
7% 10% 12,5 % 15% 5%
30% Acrylamide-bis
solution 29:1 6x SDS-buffer
(A) 2.33 3.33 4.17 5 1.67
4x Separation buffer
2 M Tris-Cl 2.4 ml
1.5 M Tris/HCl pH (pH 6.8)
8.8 2.5 2.5 2.5 2.5
4x Stacking buffer SDS 0.96 g

0.5 M Tris/HCl pH Glycerol 4.8 ml

6.8 + phenol
red 2.5
DTT 739 mg

aqua dest. 5 4 3.2 2.4 5.7 Bromophenol

4.8 mg
Blue
SDS (10 %) 0.1 0.1 0.1 0.1 0.1
TEMED 0.015 0.015 0.015 0.015 0.015
APS (10%) 0.03 0.03 0.03 0.03 0.03

78

Detection techniques
Coomassie-Blue staining: robust,
moderate sensitivity (limit ≈ 1 µg)
Silver staining: elementary silver is
deposited at the site of proteins,
very sensitive (limit ≈ 10 ng)
protein-specific fluorescent dyes:
SYPRO-Orange, SYPRO-Ruby,
Deep-Purple
(compatible with MALDI-TOF, MS)
Special stainings: Proteoglycans
(Alcian-Blau), glycoproteins
(Schiff‘s Reagent)
Autoradiographie, Fluorography

79

39
 Silver staining

66 kDa

25 kDa

separation with 12% Acrylamide: 20 - 80 kDa 80

Molecular weight assessment after SDS-PAGE

http://www.meduniwien.ac.at/user/johannes.schmid/SDS-PAGE.xls

log[MW]

migration distance starting from

stacking gel/separation gel interface
81

40
 Fluorography, Autoradiography

Sensitive detection of radioactively labeled proteins

Gel is equilibrated with a radiosensitive fluorophore:

e.g. Diphenyloxazole (POP), Sodiumsalicylate

Detection by X-ray film or Phoshor-Imager devices

82

Fluorography

Phospho-Imager detection X-ray film detection

(1 day exposure) (3 months exposure !!)

83

41
 Silver Staining of PAGE Gels
Solutions
Fixing solution: 50 % ethanol, 10 % glacial acetic acid, ad 100 % with aqua dest.
Incubating solution (1L): 30 % ethanol, sodiumthiosulfate anhydrous 2g, sodiumacetat
anhydrous 34 g, fill up to 1L with aqua dest. Before use add 125 µL of
glutaraldehyde/50 mL incubating solution.
Silvernitrate solution (1L): AgNO3 1 g, dissolved in 1L aqua dest.. Before use add 10 µL
of formaldehyde/50 mL of silver nitrate solution.
Developing solution (1L): Na2CO3 anhydrous 25 g, dissolved in 1L aqua dest.. Before
use add 10 µL of formaldehyde/50 mL of developing solution.
Stop solution (1L): sodium-EDTA 15.78 g dissolved in 1L aqua dest..
After electrophoresis, the polyacrylamide gel is taken out of the casting sandwich and
placed in a clean glass beaker filled with fixing solution. All following steps are
carried out while gently shaking. The gel has to be incubated with the fixing
solution for 30 minutes. After fixation an appropriate amount of incubating solution
including glutaraldehyde (the gel has to be at least covered by liquid) is prepared
and added to the gel, followed by incubation for 15 minutes, discarding the fixing
solution and washing with aqua dest. 3x for 5 minutes and 10 minutes incubation
in silvernitrate solution including formaldehyde. The silvernitrate solution is
collected (special waste). Developing is carried out by incubating the gel in
developing solution including formaldehyde until the desired intensity of protein
staining is reached, followed by discarding of developing solution and adding stop
solution. The gel should incubate for at least 1 hour in the stop solution.
Afterwards the gel can be stored in aqua dest. or dried with vacuum. 84

EMSA‘s (Electrophoretic mobility shift assays)

…used to monitor active transcription factors (by binding to short, labeled
oligonucleotides comprising the bound DNA sequence)

Example: comp.: competitor: non-labeled

ds-oligo of the same sequence
(usually added in > 10-fold
molar excess) – competes with
the labeled oligo for binding to
the TF > reduces the specific
signal
mut.comp.: mutated competitor:
should not compete for specific
binding

42
 Defining the composition
of TF-complexes using
antibodies and supershifts
supershift

EMSA Alternative: ABCD Assay

(Avidin-Biotin Complex with DNA)

TF

dsOligo

Biotin
Streptavidin

87

43
 Isoelectrical Focussing (IEF)

Separation of proteins
according to their isolelectric
point (pH at which they are
not charged)
Usage of immobilized pH-
gradients (Ampholines)
native IEF or denaturing IEF
(urea) can be done

88

2D-Electrophoresis

for analyses of complex protein mixtures

(→ Proteomics)
combination of IEF (1. dimension) and SDS-PAGE (2.
dimension)
high separating resolution (> 1000 Spots)
Protein pattern databases are available and software
for pattern comparison
identfication of spots by mass spectrometry or by
immunological methods (immunoblotting)

89

44
Principle of 2D-Electrophoresis

90

2D-SDS-PAGE

91

45
 2D-DIGE (Difference Gel Electrophoresis)
Protein extracts from
two different samples
are labeled with two
different fluorescent
dyes (e.g. Cy2 and Cy3)
and mixed (usually
together with a pooled
standard mixture
labeled with a 3rd dye,
eg. Cy5).
2D-PAGE is performed,
followed by scanning of
the gel with the 3
wavelengths exciting
the 3 dyes > the results
are compared by
computer analysis

Gel chromatography (size-exclusion chrom.)

• Separation technique that uses porous bead material in a
column to separate macromolecules according to size.
• Wide range of molecular weights that can be separated
• Much larger molecules can be separated than with SDS-PAGE or
native PAGE

93

46
 Gel chromatography

larger molecules are eluted first

94

Gel chromatography
Example
0.017 0.19
SERT
SERT 44 kDa 0.17
0.015 Standards
0.15
0.013
0.13
Trp-fluorescence
Ex436/Em510

0.011
0.11
158 kDa
0.009 0.09

0.07
0.007
0.05
0.005
670 kDa 0.03
0.003
0.01

0.001 -0.01
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110

min

95

47
 Spin desalting or buffer exchange based on gel
chromatography
1. Place spin column in a 1.5 mL microcentrifuge collection tube.
2. Centrifuge at 1500 x g for 1 minute to remove storage solution.
3. Add 300 μL of desired final buffer to the resin bed and centrifuge at 1500 x g for 1 minute;
discard flow-through
4. Place the equilibrated spin column into a new collection tube
5. Load your protein sample (130 µl)
6. Centrifuge at 1500 x g for 2 minutes to collect desalted sample.
7. Protein sample is now desalted & buffer exchanged and ready for use

96

Immunoblotting

Western Blotting: Electrophoretic protein-transfer

from a gel (mostly SDS-PAGE) onto a membrane

Membranes: Nitrocellulose,
Polyvinyl-difluorid (PVDF)

detection via enzyme-coupled antibodies

Enzymes: - Horse Radish Peroxidase (HRP)

- alkaline phosphatase (AP) 97

48
Immunoblotting II

98

Wet Blotting
equipment

99

49
 Blotting conditions

100

Semidry blotting (BioRad)

• wet Millipore Immobilone-P

membrane in MeOH, rinse with
water, then 1 min. in Blotting buffer:
25mM Tris Base
150 mM glycine
10% methanol
(make a 90% stock without MeOH,
which is then added freshly)
filter 0.2 mm !!!
• wet 5 3MM filter papers -> anode
• then wetted membrane
• then gel
• then 5 wetted filter papers
• wet cathode, close lid quickly
without moving back and forth.
• blot 250 mA for a small Bio-Rad gel
(should be 20, increasing to 30 V)
for 30 min.

101

50
 Immunoblotting: various substrates

Chromogenic
Chemiluminescence
detection of HRP -substrate

102

Western Blot Protocol

• Detection:
• block 1 hour to O/N in PBS/5% milk at RT
• wash 3x PBS/0.1%Tween 20
• 1st AB in PBS/Tween, 1 h
• wash 3x 5 min PBS/0.1%Tween 20

• add 2nd AB in PBS/Tween (e.g., peroxidase-conjugated donkey

anti-rabbit Amersham, at 1:10.000) for 1 hour
• wash 3x 5 min
ECL-system:
• combine equal volumes of soln. A and soln. B (Amersham ECL or
Pierce SuperSignal WestPico)
• add to filter, incubate 1 min. (Amersham), or 5 min (Pierce)
• roll away, cover with saran wrap, expose within 10 min.
• (exposure times can vary between seconds and 20 min.)

103

51
 ECL reagent: selfmade version

Advantages: almost no cost; stable/reproducible, stored in frozen aliquots

To 10 mls of 100 mM Tris pH 8.5 (RT), add
50 µl luminol (warm to redissolve)
22 µl of coumaric acid (warm to redissolve)
3 µl of H2O2 (fresh < 6 months)
Pour onto blot for 1 minute and process as normal (10 ml enough for 100
cm2)
Stock luminol: 250 mM 3-aminopthalhydrazide (Fluka #09253); 266
mgs in 6 mls DMSO; store frozen in 60 µl aliquots.
Stock coumaric acid: 90 mM coumaric acid (Sigma C9008): 38 mgs in
2.5 mls DMSO; store frozen in 25 µl aliquots.

STRIPPING of membranes: with 2% SDS, 62.5mM TRIS pH6.8,

100mM Beta-mercaptoethanol for 30 min at 50°C
104

Immunoprecipitation
Antigen

Interaction between antibody and

antigen in solution
Isolation of immuno-complexes by
Protein A- Agarose (Sepharose) – Beads
Protein A = IgG-binding protein of
Staphylococcus aureus
Alternative: Protein G: often used for
mouse monoclonal antibodies, which are not
well bound by Protein A

- or antibody covalently linked to activated

Sepharose (CNBr-activated)
Analysis by SDS-PAGE

105

52
 Example of an Immunoprecipitation

Proteins labeled unspecifically with 35S-methionine > specific detection of a

protein of interest by immunoprecipitation > fluorography

106

Affinity chromatography

• beads can be coupled

with antibodies or other
affinity ligands, which
bind a molecule of
interest.
• after washing of the
beads, the captured
target molecules can be
eluted e.g. by lower pH.

107

53
 Analysis of post-translational
modifications

proteolytic processing
Glycosylation
Phosphorylation
Ubiquitination
….

108

Analysis of proteolytic processing

in vivo: cultivation of cells in presence of selective

proteinase inhibitors
inhibition of intracellular proteinase (in cytosol, ER,
Golgi) is possible just with membrane permeable
inhibitors
inhibitors that are not membrane permeable act just
in the extracellular environment, in endosomes and
lysosomes
in vitro: Incubation with specific proteinases

109

54
 Proteolytic processing (in vivo)

Z-FA-CHN2

Z-FA-CHN2 .... Benzyloxycarbonyl-Phenylalanyl-Alanyl- 110

Diazomethane (inhibits cysteine proteinases)

Detection of N-Glycosylation

in vivo: Biosynthesis in presence of inhibitors

Tunicamycin: inhibits the initiation of N-Glycosylation
Processing of N-Glycans blocked by Glycosidase-
Inhibitors

Tunicamycin (µg/ml) 0 0.1 0.5

111

55
 Analysis of Glycosylation (in vitro)

Endo H .... Endoglucosaminidase H; cleaves just

Mannose-rich N-Glycans
PNGase ... Peptid:N-Glycosidase F ("N-Glycanase") 112

Analysis of Phosphorylation

in vivo: metabolic labelling with

[32P]Phosphate (e.g. in presence or
absence of an expression or suppression
construct for a specific kinase, followed by
immunoprecipitation)
in vitro:
- Incubation with alkaline Phosphatase
- Phospho-aminoacid analysis (DC)
- Kinase-Assays
Immunoblotting/Immunprecipitation
(e.g. with anti-phospho- specific Antibody
or for instance anti-Phosphotyrosin)

113

56
 In vitro Phosphorylation Analysis

PNGase ... Peptid:N-Glykosidase F ("N-Glykanase")

AlkPhos ... Alkaline Phosphatase
114

in vitro Kinase Assay

1. Immunoprecipitation of the kinase
2. Kinase-Reaction in presence of 32P-γ-ATP

3. SDS-PAGE and Fluorography

32P-γATP +
substrate phosphorylated substrate

Immunoprecipitated Kinase

antibody
Agarose-Bead

115

57
 Kinase Assay- Protocol
Lysis buffer (final conc.): for 20 ml:
20 mM Tris/HCl pH7.5400 µl 1 M150 mM NaCl600 µl 5 M25 mM b-glycerophosphate500 µl 1 M2 mM EDTA80 µl 0.5 M2 mM
pyrophosphate400 µl 0.1 M1 mM orthovanadate200 µl 0.1 M1% Triton X-1002 ml 10%1 mM DTT20 µl 1 M1 mM
NaF20 µl 1 MA. dest.15.8 ml
Protease Inhibitors: added before use (Leupeptin, Pepstatin, Pefa-Block) according to stock
Kinase buffer (final conc.): for 20 ml:
20 mM Tris/HCl pH7.5400 µl 1 M20 mM b-glycerophosphate400 µl 1 M100 µM orthovanadate20 µl 0.1 M10 mM
MgCl2200 µl 1 M50 mM NaCl200 µl 5 M1 mM DTT20 µl 1 M50 µM ATP50 µl 20 mM1 mM NaF20 µl 1 MA. dest.18.7
ml
Lyse cells (in 6 wells) with 500 µl per well of Lysis buffer (+ protease inhibitors):
20 min at 4°C.
Clear by centrifugation (14000 rpm, 4°C 15 min Eppendorf centrifuge). Save an aliquot (30 µl) for Western blotting.
Immunoprecipitate the kinase (e.g. with 10 µl anti-flag affinity matrix beads, Sigma, for flag-tagged transfected kinase; or
with appropriate antibody for endogenous kinase + Protein A-Sepharose or directly coupled to agarose): 2h at 4°C
(rotating).
Wash the beads: 3x with 1 ml PBS (4°C), 1x with 1 ml Kinase buffer (4°C): pellet the beads by centrifugation (14000 rpm,
4°C, 45sec) and remove the supernatant.
Prepare Kinase buffer: add MnCl2 to 10 mM (stock: 1 M) and 32P-g-ATP (5 µCi per sample, usually 1/10 volume, i.e. 1 µl
of stock solution for one 10 µl assay) and preincubate at 30°C for 10 min.
Add 1 µg substrate: GST-IkB (1 µl) or mutant substrate (as control) to the beads; add 10 µl kinase buffer, mix gently and
incubate at 30°C for 30 min (or longer).
Stop the reaction by addition of 4x SDS-sample buffer (4 µl) and perform SDS-PAGE with the samples, followed by fixation
of the gel (10% methanol, 10% HAc), drying and autoradiography.
For detection with PhastGel: use only 5 µl beads, 5 µl kinase buffer, 0.5 µl substrate and 2 µl 4x SDS-sample buffer:
116Run a
12.5% PhastGel with 4 µl per sample

Detection of Ubiquitination
Potential set-up:

• transfection of a tagged ubiquitin

(e.g. His-tagged) together with the
gene of interest (e.g. flag-tagged)
• immunoprecipitation of the gene
of interest
• optional: resuspend and heat the
beads in 1% SDS-buffer, dilute to
0.05% SDS and repeat
immunoprecipitation to get rid of
potential co-precipitating,
interacting proteins.
• resuspend and heat the beads in
SDS-PAGE buffer
• SDS-PAGE
• Western Blot for the His-tag

58
 Subcellular Fractionation
(Separation of subcellular compartments)

most often used: density gradient centrifugation

coarse separation: differential centrifugation

Detection of subcellular compartments by specific

markers (enzymes that are nearly exclusively in that
compartment)

118

Differential centrifugation

Subsequent centrifugation steps with increasing g-force

119

59
 Gradient centrifugation:

Samples are usually layed on top of a gradient, proteins

or compartments migrate through the gradient in „zones“

120

Density gradient centrifugation

Gradients: continuous ↔ discontinuous („steps")

self-forming gradients (e.g. Percoll)
(density equilibrium centrifugation)
samples are either layered on top of the gradient or at the
bottom
Fractionation after the centrifugation (e.g. by peristatic
pump and fraction collector)

gradient mixer: B A 0
100
A
B
100
0
volume (or time) 121
Magnetic stirrer

60
 Substances to generate density gradients

Sucrose: low molecular weight (342 Da),

osmotically active

Ficoll: copolymerisate of Sucrose and

Epichlorhydrine; Mr ≈ 400 000 Da

Percoll: colloidal silica gel

special case for DNA: Cesiumchloride

122

Self-forming gradients (e.g. Percoll..)

123

61
 Example for a density gradient centrifugation

Separation of lysosomes ( ) and Golgi ( )

in a continuous Percoll gradient (density º) 124

Research Methods - Overview

• cell culture systems

• labelling and transfection of cells
• analyses of cellular components
• analyses of molecular interactions
• fluorescence measurements
• microscopy
• flow analysis (FACS)
• analyses of cellular processes (proliferation, apoptosis..)

125

62
Methods to investigate macromolecular interactions

• Interaction screening with phages (Phage Display)

• Yeast 1-Hybrid System (protein : DNA)
• Yeast 2-Hybrid System (protein : protein)
• Mammalian 2-Hybrid System
• Gel-Chromatography
• Co-Immunoprecipitation
• Fluorescence Resonance Energy Transfer (FRET)
– see fluorescence methods

126

Interaction screening with phages

(Phage Display)

A gene library is expressed on the

surface of appropriate phages (e.g.
M13), which are incubated with specific
target proteins immobilized on plates.
Unbound phages are washed off; bound
phages are eluted by lowering the pH.
3-4 x

Bound phages are amplified and again

incubated with plates containing the target
proteins – this repeated 3 – 4x to enrich the
specifically binding phages. Clones are isolated
and sequenced > Sequence of the binding
protein
127

63
 Yeast 1-Hybrid System
For the identification of proteins that bind specifically to a given DNA sequence (e.g.
transcription factors; DNA:protein interaction).
The DNA sequence of interest (e.g. from a promoter) is usually cloned in repeats (3-5x)
in front of an appropriate selection gene (e.g. a histidine synthesis gene) and an
appropriate reporter yeast strain (which is not capable of growing in the absence of
histine) is stably transformed with this construct. Subsequently, this yeast strain is
transformed with a library containing putative binding proteins (often fused to the
transactivation domain of the Gal4 transcription factor). Binding of a protein to the DNA
sequence results in growth of this yeast clone on selection plates.

Gal4-Activation domain
Transcription > growth on
Insert from library selection plates

TATA HIS lacZ

DNA-Region with potential protein
binding sites (in repeats)
128

Yeast 2-Hybrid System

A yeast strain is used, which does not contain a functional Gal4 transcription factor –
but reporter and selection genes, which are downstream of Gal4-dependent promoters
(Histidine- and Adenine-synthesis genes, lacZ for β-Galactosidase expression, which
can be used for staining). This strain is transformed with putative interaction partners:

1. fusion protein of the Gal4-DNA-binding

domain and Protein X („bait“)

2. fusion protein of the Gal4-

transactivation domain and Protein Y (or a
library insert; = „prey“)

Co-Transformation or combination by
yeast mating

> In case of an interaction between protein X

and Y auftritt, a functional transcription factor is
build, which binds to Gal4 promoters – and the
cells can grow on selection plates (without His
or Ade).
129

64
 Preparation of a Yeast 2-Hybrid Screen

1. Cloning of the gene of interest into the bait

vector“ (in frame with the Gal4-DNA-
binding domain): selection in bacteria (e.g.
via Kanamycin), selection in yeast (e.g. via
Trp-synthesis gene))
2. Test for Auto-Activation with „empty
Gal4AD-vector): Tests whether the gene of
interest interacts with the Gal4 activation
domain (without the need of protein Y):
If it does so, the bait cannot be used in the
yeast 2-hybrid system.

The Gal4 AD vector contains a second

selection gene for yeast (e.g. leucine
synthesis gene). Co-transformants of the
two vectors grow in the absence of Trp and
Leu; but they should not grow in the
absence of the amino acid that is
synthesized just when an interaction occurs
(e.g. in the absence of histidine or
adenine). 130

Combination of the two putative interaction partners

Reporter strain Reporter strain Y187
(e.g. AH109) pretransformed with a library (in
Mating Type: a Gal4AD-Vector), Mating Type: α

bait
X Gal4AD
Gal4BD

Mating: Incubation of the two haploid Instead of mating the two vectors can be
strains for 24 h at 30°C, 40 rpm combined by classical transformation
> formation of diploid clones with both
vectors
clones, which contain interaction X
His, Ade,
partners grow on selection plates and lacZ
express lacZ

PCR from single colonies (with primers specific for the library vector)
purification of PCR-Products

sequencing of the putative

interaction partners 131

65
 Example of a yeast two-hybrid result

B) Streaking out the colonies from the

A) single colonies on selection first selection plates to secondary
plates (SD-Leu-Trp-Ade) selection plates (e.g. with higher
selection pressure and stringency:
SD-Leu-Trp-Ade-His)

132

Verification of a yeast 2-hybrid result

1. Analysis of the sequence and comparison with database: check whether the ORF
is OK (in frame with the Gal4AD)
2. Isolation of Plasmid-DNA from the yeast colony
3. Re-transformation in E.coli (to separate bait and prey – using different antibiotics
resistance) and preparation of the plasmid containing the library insert
4. „False Positive Test“ in yeast: Transformation of the Gal4AD-plasmid containing
the identified „prey“ with the empty Gal4-binding domain vector: this shouzld not
lead to growth on selection plates of interaction (if there is growth, then the
library insert interacts with the Gal4BD and not the bait protein)
5. β-Galactosidase-assays (also quantitative, to compare interaction partners)
6. Verification in the correct cells (human cells), e.g. by co-immunoprecipitation

133

66
 Example of a “False-Positive Test”

1,7

1,6

1,5
rel. activity

1,4 Example of lacZ

1,3
quantification
1,2

1,1

1
neg. control IKK2/GMRa IKK2/GMRb 134

LiAc Yeast Transformation

Solutions:
• Synthetic drop out solution 10 x in AD: L-isoleucine 300 mg/L, L-valine 1.5 g/L, L-adenine hemisulfate salt 200
mg/L, L-arginine HCl 200 mg/L, L-histidin HCl monohydrate 200 mg/L, L-leucine 1 g/L, L-lysine HCl 300 mg/L, L-
methionine 200 mg/L, L-phenylalanine 500 mg/L, L-threonine 2 g/L, L-tryptophan 200, L-tyrosine 300 mg/L, L-
uracil 200 mg/L
• SD -Trp medium (synthetic dropout medium): synthetic minimal medium lacking tryptophan: yeast nitrogen
base without amino acids 6.7 g/L, 2 % dextrose (glucose) (sterile dextrose solution is added after autoclaving
to avoid maillard reactions), pH adjusted to 5.8, for plates : agar 1.5 g/L
• YPD (yeast peptone dextrose) broth, yeast complete medium: yeast extract 10 g/L, peptone 20 g/L, 2 %
dextrose (glucose), pH adjusted to 5.8 DNA + water + carrier-DNA or RNA
• Aqua dest. sterile 1M LiAc
• LiAc 100 mM sterile
• LiAc 1 M sterile Polyethylenglycol
• Bacterial RNA, used as carrier
yeast pellet
• Poly-ethyleneglycol PEG 50 % (w/v) sterile filtered
10 mL of SD -Trp medium are inoculated with the appropriate yeast strain and incubated at 30°C while shaking at 200
rpm o/n. On the next day OD at 600 nm is measured and the yeast culture is diluted with YPD to OD600 0.1. A
total volume of 50 mL diluted yeast culture is used for further incubation. Every hour OD600 is measured until
OD600 0.4 is reached (3 - 5 hours). Then the cell number is calculated with a Thoma chamber. 2x107 cells/mL
are sufficient for 10 transformations. The yeast is then harvested by centrifugation at 3000 rpm for 5 minutes,
the supernatant is carefully removed and collected for autoclaving. The pellet is resuspended in 25 mL sterile
AD and again centrifuged at 3000 rpm for 5 minutes. After removing of the supernatant the pellet is
resuspended in 1 mL LiAc 100 mM. Excess of LiAc is removed by spinning the tubes for 15 seconds at full speed
in a tabletop centrifuge and carefully removing the supernatant. The yeast pellet is brought to a final volume of
500 µL with LiAc 100 mM. Aliquots of 50 µL are prepared. One 50 µL aliquot of this yeast suspension is used for
one transformation. 50 µL aliquots are again briefly centrifuged to pellet the cells, the supernatant is removed
and on top of the yeast pellet, layers of the following transformation solutions are pipetted in following order:
240 µL 50 % PEG, 36 µL LiAc 1 M, 3.3 µL of bacterial RNA (31 µg/µL), 70.7 µL sterile AD, 1 µg plasmid DNA
(1µg/µL). The tube is then thoroughly mixed by vortexing for 1 minute until the yeast pellet is completely
dissolved and placed for 30 minutes in a 30°C water bath. The tube is then transferred to a 42°C water bath for
25 minutes in order to perform the heatshock. The transformation mix is then briefly centrifuged for 15 seconds
at 4 000 x g (7 000 rpm) in a table top centrifuge, the supernatant is discarded and the pellet is resuspended135in
1 mL sterile AD. 50 µL of this transformed yeast suspension are plated on SD – Leu, - Trp, - Ade plates and
incubated at 30°C for some days.

67
 Mammalian 2-Hybrid System

Posttranslational modifications such as

phosphorylations, which might be
essential for interactions are often not
carried out in yeast. In this case a
similar assay can be set up in
mammalian cells (e.g. providing the
kinase)
Limitations:
1. not suited for screening
purposes
2. Proteins are in the nucleus
and thus eventually not at
their normal localization

> FRET-Microscopy: as alternative to

visualize protein-protein-interaktion in
their physiological context
136

Example for a mammalian 2-Hybrid Test

137

68
 Biochemical Verification of Protein-Interactions
by Co-Immunoprecipitation (CoIP)

1. The 2 proteins of interest are transfected into mammalian cells (usually containing
to different tags, e.g. HA- and flag). 1 – 2 d after transfection, the cells are lysed
and one protein is immunoprecipitated using antibody-beads against tag1 (e.g.
flag). The beads are washed extensively with buffer (isotonic or hypertonic, not
hypotonic) and finally heated with SDS-buffer to release bound proteins. SDS-
PAGE and Western blotting is performed – using antibodies against tag1 and
against tag2. If protein with tag2 co-precipitated with protein containing tag1, then
there is interaction.
2. Co-immunoprecipitation of endogenous proteins (without transfection) – using the
same principle and antibodies against the endogenous proteins

1. Protein 2. 3.
Protein Y-HA HRP
X-myx
Antikörper

bead

138

Example for a Co-Immunoprecipitation

1. Co-IP with overexpressed proteins 2. Co-IP with endogenous proteins
(after transfection); control: control: IP with unrelated antibody
transfection with just one protein

139

69
 The salt concentration has an influence on the
stringency of the co-immunoprecipitation
Co-Immunoprecipitation of TRAF1 and
TRAF2 at 500 mM NaCl
Co-Immunoprecipitation of TRAF1 und
IKK2 works at 250 mM NaCl, but not at
500 mM > TRAF1/TRAF2 interaction is
stronger than TRAF1/IKK2 interaction

140

Co-Immunoprecipitation for the Detection of Protein Interactions

1. Transfection of cells with tagged proteins (one 6-well of CHO or HeLa cells is sufficient for one sample).
2. Preparation of extracts:
2.1. 1 d after transfection: wash cells with PBS
2.2. Lysis with 500 µl/well Lysis-Buffer + Protease Inhibitors: 15 min at 4°C.
Buffer: 0.5% NP40, 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 150 mM NaCl.
Protease Inhibitors: 10 µg/ml Aprotinin, 20 µg/ml Phosphoramidon, 40 µg/ml Pefabloc, 1 µg/ml Leupeptin, 1 µg/ml
Pepstatin (from 1000x stock solutions, Boehringer Protease Inhibitor set).The lysis is suited for cytosolic proteins and
membrane proteins. Nuclei remain intact (you can leave the nuclei on the plate when you take off the supernatant).
2.3. Spin the extracts for 15 – 30 min at 14 krpm, 4°C (HeLas: 15 min, CHO: 30 min)
2.4. Keep the supernatant and adjust the NaCl-concentration (150 mM – 1000 mM depending on the strength of
interaction; start in the range of 150 – 250 mM, increase the concentration if you want to increase the stringency)
3. Co-Immunoprecipitation
3.1. Take 400 µl of extract for IP (keep about 30 µl extract for direct western analysis).
Use flat-top tubes (the visibility of the pellet is better in these tubes) Add 400 µl Lysis-Buffer/250 mM NaCl (without
NP40 > final concentration: 0.25%). Add beads (15 µl anti-flag-M2-Agarose, Sigma A-1205; alternatives: other
antibodies directly coupled to CNBr-activated Sepharose; Protein A- or Protein G-Agarose: the later will give more
unspecific binding). Rotate extracts + beads for 2 h at 4°C.
3.2. Spin for 30 sec at 14 krpm 4°C. Take off the supernatant, add 1 ml of lysis buffer/250 mM NaCl/without NP40
and invert tubes several times (do not vortex). Repeat this washing step.
3.3. Suspend the beads in 1 ml cold PBS and transfer the suspension to a new tube.
Spin 30 sec at 14 krpm, 4°C, take off the supernatant and repeat this washing step. Final centrifugation: 1 min at 14
krpm, 4°C. Remove the supernatant and suspend the beads in SDS-PAGE buffer (30 µl). Incubate for 5 min at 95°C
and pellet the beads for 2 min at 14 krpm.
4. SDS-PAGE
5. Western Blot: if possible use HRP-conjugated primary antibodies (anti-HA-HRP from Boehringer, anti-myc-
HRP from Invitrogen). This gives much lower background of unspecific bands (Ig light chain …). 141

70
 Far Western Blotting

… the membrane is probed with a protein, which can bind the protein of
interest. While western blotting detects certain proteins using antibodies, far-
western blotting detects protein:protein interactions.

Research Methods - Overview

• cell culture systems

• labelling and transfection of cells
• analyses of cellular components
• analyses of molecular interactions
• fluorescence measurements
• microscopy
• flow analysis (FACS)
• analyses of cellular processes (proliferation, apoptosis..)

143

71
 Principle of Fluorescence
1. electrons of a fluorophore are
excited by absorption of an
appropriate photon (hν −Ex) and
their energy state is raised to S1´
2. the excitated state S1´exists for
about 1 – 10 nsec. Energy is lost
by several reactions
Jablonski-Graph (interaction…) leading to the
excited state S1.

3. Electrons fall back from S1 to S0 – the energy difference is

released by emission of a photon (which has lower energy than the
excitation photon – and thus a longer wavelength) according to
λ = c/ν
144

Principles of Fluorescence
double bonds = flexible (delocalized) p-electron system

from:
http://www.invitrogen.com/site/us/en/home/support/Tutorials.html

72
delocalized p-electron systems
(alternating double bonds) can
easily absorb photons and
thereby be raised to higher
energy levels

delocalized p-electron systems

(alternating double bonds) can
easily absorb photons and
thereby be raised to higher
energy levels

73
 energy loss due to
movements, rotations etc...

... sudden fall from an excited

energy level to the ground
state

74
 The absorbance of light
(photons) depends on the
colour (the wavelength)
number of
absorbed
photons

excitation spectrum

75
 The emitted light (photons)
exhibits a certain wavelength
spectrum (colour) –
depending on the nature of
the fluorophore

number and colour of

emitted photons

emission spectrum

153

76
 Excitation and Emission Spectra

Stoke‘s Shift

Infos: http://www.probes.com/servlets/spectra/
Java-Applet from BD: http://www.bdbiosciences.com/spectra/
154

Java-Applet from BD:

http://www.bdbiosciences.com/spectra/

155

77
 Characteristics of fluorescent dyes

Excitation Maximum: wavelength of maximal photon

absorbance (λ in nm)
Emission Maximum: wavelength of maximal photon
emission (fluorescence, λ in nm)
Molar Extinction coefficient: gives the absorbance of
excitation photons at the excitation maximum λ (in cm-
1M-1)

Quantum-Yield: number of emitted photons per

number of absorbed photons.

Brightness = molar extinction coeff. x quantum yield

156

Parameters of some important fluorescent dyes

dye Ex Em DAPI... 4’,6-Diamidino-2-Phenylindol

DAPI 359 461 FITC... Fluorescein Isothiocyanat

TMRho...Tetramethylrhodamine
FITC 494 520
(TRITC: Tetramethylrhodamine Isothiocyanate)
TMRho 550 573
TexasRed 595 615
157

78
 Alexa-Fluorophores from Molecular Probes/Invitrogen
(www.invitrogen.com)

158

GFP (Green Fluorescent Protein) and its variants

• Structure: barrel like with the chromophore in

the middle
• MW: appox. 29 kDa
• original protein from jellyfish (Aequorea
victoria), exists in bacterial and mammalian
codon optimized versions.
• Point mutations were incorporated improving
the fluorescence (enhanced GFP: EGFP) and
also leading to other spectral variants (colours;
ECFP, EYFP…)
• Fluorescent in living cells – can be expressed
as fusion protein with the protein of interest (is
usually not altering the function of the target
protein)

159

79
 Fluorescence Properties of some GFP-Variants
Variant Excitation (nm) Emission (nm)
EBFP (Blue) 380 440

ECFP (Cyan) 433 475

EGFP (Green) 488 507

EYFP (Yellow) 513 527

DsRed 558 583

160

Other fluorescent proteins

fluor. protein Ex-Peak Em-Peak quantum yield comment
nm nm
EBFP 380 440 0.18 Clontech

ECFP 433 (453) 475 (501) 0.4 Clontech

EGFP 488 507 0.6 Clontech

wildtype GFP 397 (475) 509 0.77 Aequorea victoria

EYFP 513 527 0.61 Clontech

Citrine 516 529 0.76 Griesbeck et al. 2001

DsRed 558 583 0.29 Clontech, tetramer

DsRed2 0.55 tetramer,

563 582
HcRed1 588 618 0.02 Clontech, dimer

PA-GFP 400 before act. 515 before act. 0.13 photoactivatable GFP, T203H
(Patterson 504 (397) 517 after act. 0.79 mutant of mammalian
after codon-optimized wildtype
2002)
GFP
PS-CFP 400 before act. 468 before act. 0.2 photoswitchable CFP, turns from
490 after act. 511 after act. 0.23 cyan to green after intense
illum. at 405 nm
mOrange 548 562 0.69 Shaner et al., 2004

mStrawberry 574 596 0.29 Shaner et al., 2004

mCherry 587 610 0.22 Shaner et al., 2004 161

dTomato 554 581 0.69 Shaner et al., 2004, dimeric

80
 Photoconvertible fluorescent proteins
mOrange conversion to far-red
(2x bleaching with 100% 488 nm in between)
120

100

80
rel fluor

60 mOrange
converted far red
40
control cell
20

0
0 20 40 60 80 100 120
sec

mOrange

Far-Red

Photo-switchable fluor. protein Dronpa

120 start bleaching at 488 nm

100

80
% of initial fluor.

60 start reactivation
at 350 nm

40

20

0
-20 0 20 40 60 80 100 120 140 160 180 200
sec

81
 Fluorimetric Analysis Methods

Fluorescence measurements are usually more sensitive than

photometric measurements.
Scanning fluorometers have usually 2 monochromators for adjusting
excitation and emission wavelengths. Most instruments also allow to
adjust the bandwidth of excitation and emission (between 1–20 nm)
The emitted fluorescence is measured by a photomultiplier tube
(PMT) detector. The sensitivity of that can be adjusted by changing
the voltage (e.g. 400 - 700 V).

Emission

Detector
Monochromators 164

Parameters of fluorometry

• Excitation wavelength in nm
• bandwidth of the excitation light (1 – 20 nm, „slit width“)
• Emission wavelength in nm
• bandwidth of the emission
• sensitivity of the detector („gain“, voltage of the PMT)
• Integration time of the measurement (slow – fast, in sec.:
influences the „noise“)

165

82
 Wavelength Scans
The exact emission and
excitation peaks might differ
Emission spectrum (ECFP) slightly between different
2
fluorometers.
For checking the parameters:
1.5 - run an excitation wavelength
scan at the literature value of the
peak emission
rel. fluor.

1 - run an emission scan at the

determined excitation peak
- repeat the excitation scan at
0.5
the determined emission peak
For adjusting these parameters
you have to consider the
0 fluorescence properties (e.g. the
460 480 500 520 540 560 580
Stoke‘s shift) – to prevent that
nm
excitation light is detected
166

Tricks for optimizing fluorescence

measurements based on the spectra

excitation possible emission

window window

Fluor.
real
emission
curve 1

theor. spill-over of the excitation light

excitation theor.
curve emission
curve

detected fluorescence

nm

83
 Tricks for optimizing fluorescence
measurements based on the spectra

excitation
window
more
narrow
Fluor.
emission window

theor.
excitation emission curve 2
curve

detected fluorescence

nm

Tricks for optimizing fluorescence

measurements based on the spectra

left shifted
broader excitation
window

Fluor.
real
emission
curve 1
real emission curve 3

detected fluorescence

nm

84
 Time Scans
Can be applied to determine the time course of fluorescence changes (e.g. to
determine enzyme reaction kinetics – or for instance in chromatography to
measure the kinetics of elution and thus the molecular weight of a fluorescent
compound, such as a GFP-fusion protein)

0.017 0.19
SERT
Standards SERT 44 kDa 0.17
0.015
0.15
0.013
0.13

Trp-fluorescence
Ex436/Em510

0.011
0.11
158 kDa
0.009 0.09

0.07
0.007
0.05
0.005
670 kDa 0.03
0.003
0.01

0.001 -0.01
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
min
170

Fast Kinetic-Analysis
(Stopped-Flow Fluorometry)

Two reaction partners are injected into a mixing chamber, where they are mixed
within approx. 1 msec by stopping the flow.
If the reaction between the two compounds changes the fluorescence, this change
can be recorded with a resolution in the microsecond range.

Light-Absorbance
(Stopped Flow Photometry)

A
Fluorescence
(Stopped Flow Fluorometry)
B

Excitation light 171

85
 Example for a stopped-flow fluorometry

Tangent of the initial fluorescence change (reaction kinetics rate in 1/sec)

Linear correlation between the initial

0.008 reaction kinetics range and the
0.007 Tet-DNA concentration of the reaction
rel. fluorescence

0.006 partners
0.005 kon koff
0.004 y = 734,21x + 244,09
0.003 900 R2 = 0,9508
0.002 mutant Tet-DNA 800 rate (15°C)
0.001
700 rate (37°C)
0.000

rate (1/sec)
600
-0.001
-0.002 500 kon
0 0.02 0.04 0.06 400
300
seconds
200
100 koff
0
0 0,2 0,4 0,6 0,8
Kaff = 1/Kdiss = kon / koff DNA (µM)
172

Quantitative Fluorometry

Fluorescence measurements can be used to quantify a great variety of

different substances.
Usually a standard curve is measured with the optimized measurement
parameters (e.g. after defining them by wavelength scans: excitation
and emission wavelengths and corresponding bandwidths; PMT voltage
and integration time).

Some examples for quantitative fluorometry

1. Enzyme-Measurements (e.g. β-Galactosidase)

2. DNA-Measurements (Hoechst 33258, SYBR Green): fluorescent
dyes, which intercalate into the DNA and are fluorescent
dependent on the amount of DNA
3. Protein-Measurements (inherent fluorescence due to aromatic
amino acids such as Tryptophane (dye: SYBR Orange, …)

173

86
 Example for quantitative fluorimetric
measurement

7
Standard curve
6
Probe
Fluorescence (Em 376/ Ex 276)

y = 0.8738x + 0.1417
1 2
R = 0.9959

0
0 1 2 3 4 5 6 7 8
µg/ml

174

Fluorescence as measurement value in special

analysis techniques: Real-Time PCR
In real: samples contain different
amounts of cDNA, but this difference
Conventional PCR: is not detected, when they reach the
same plateau at the end of the
Comparison of a gene of
reaction > this can be revealed by
interest with a housekeeping
measuring the reaction product (by
gene using an endpoint
fluorescence) after each cycle
determination

Sample 1 2

1
endpoint
2

175

87
PCR Principle

176

177

88
 178

Scheme of a qPCR machine

LightCyclerTM, Roche heating

Ventilator

capillary with
the PCR-mix

dichroic mirrors

threshold

Ct
light source detectors
179

89
 Realtime PCR machines
Applied Biosystems
Roche Light Cycler
StepOne Plus

capillaries
(app. 1 €/sample)

96-well plates

180

Real-Time PCR with SYBR Green as DNA-

fluorescence dye
SYBR Green intercalates in the
amplified dsDNA (PCR-product)-
leading to an increase in fluorescence
with increasing cycle number.

After many cycles the fluorescence

also increases in the water control –
due to the formation of primer
aggregates

181

90
 Melting point analysis of the PCR-product

Specific and unspecific PCR

products can be distinguished
by their different melting
temperature (as determined
after the PCR by slow heating
and the decrease of the
fluorescence at the melting
point). This can also be applied
to detect mutations.

measurement of the fluor. after each cycle at a

temperature above the melting temperature of the
182
unspecific PCR product allows quantifying just the
specific product

Real-Time PCR with FRET-Hybridisation Probes

Within the sequence flanked by Primer 1 and
Primer 2 (amplification primers), two additional
oligonucleotides (Hybridisation Probes 1 and 2)
are situated, which contain two different
fluorophores at the 3‘ and 5‘ ends. When these
oligos bind to the PCR product, the fluorophores
come into close proximity and one fluorophore
can transfer part of its fluorescence energy to the
other one (fluorescence resonance energy
transfer, FRET), which then starts to shine. In
this case primer aggregates do not generate a
fluorescence signal.

183

91
 Real-Time PCR with TaqMan probes
A TaqMan-probe contains FRET-Donor and Acceptor (Quencher)-Fluorophore within the
same oligonucleotide. At the annealing temperature of the oligo, the probe binds to the PCR
product. The exonuclease activity of the Taq-Polymerase cleaves the probe and leads to
increase of the Donor-fluorescence due to de-quenching.

184

Realtime PCR – Quantification of gene

up/down-regulation
1. Determine the PCR efficiency of your PCR efficiency

gene of interest and that of your 35

housekeeping (reference) gene using 33
y = -1.67ln(x) + 25.767
R² = 0.9972
31
serial dilutions (e.g. of plasmids or 29
cDNA preparations): E = ideally 2 27
crossing point CP
CP

25
(duplication at each cycle) but 23
Log. (crossing point
CP)

realistically lower (e.g. 1.8) 21

19
17
15
0.1 1 10 100
ng cDNA input
2. Calculate up- or downregulation of
your specific gene of interest using
the differences in the crossing point ∆∆Ct-method
(CP) values with the equation: (Pfaffl MW: A new mathematical model for
relative quantification in real-time RT-PCR.
Nucl Acids Res 2001, 29(9):e45
∆CPt arg et ( control − sample )
( Et arg et )
ratio = ∆CPref ( control − sample )
( Eref )
(Excel template on my website)

92
 100000
Calculating the PCR
10000 efficiency from the
shape of the curve
log (Fluor)

1000

100
- Without using a dilution curve
10 - Can be calculated for each
0 10 20 30 40 50
sample separately
cycle

80000

70000 Neurosci Lett. 2003 Mar 13;339(1):62-6.

60000 Assumption-free analysis of quantitative
50000
real-time polymerase chain reaction (PCR)
data.
40000
Fluor

Ramakers C, Ruijter JM, Deprez RH,

30000 Moorman AF.
20000

10000

0 (Excel template on my website)

0 10 20 30 40 50
-10000 186
cycle

LinRegPCR Software
http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR

187

93
 188

Research Methods - Overview

• cell culture systems

• labelling and transfection of cells
• analyses of cellular components
• analyses of molecular interactions
• fluorescence measurements
• microscopy
• flow analysis (FACS)
• analyses of cellular processes (proliferation, apoptosis..)

189

94
 Microscopy: Human vision and the concept of
magnification

image formation in the human eye

2-step magnification principle of a

microscope with 2 lenses:
objective and eye piece (occular)

190

Basics of optical resolution I

Fine structures induce a diffraction of light (light

of zero-order, 1st order ...). Light diffraction on a
small iris is more or less equal to diffraction on
small cellular structures:

sinθ(1) = 1.22(λ/d)

θ ... angle to the first light minimum

λ... wavelength
d ... diameter of the iris

for very small angles θ: θ(1) ≅ 1.22(λ/d)

objects that are closer than θ(1) cannot be resolved as
separate objects

191

95
 Basics of optical resolution II
The more orders of light are resolved the better is the resolution.
The optical resolution that can be achieved is defined by the so called
numerical Aperture (N.A.) of the objective.
N.A. = i sin q
i ... Refraction index of the medium
(e.g. 1.0 for air, up to 1.56 for oil)
q... half of the objective opening angle
(Aperture)

192

„Airy“ disks: optical basic structures

193

96
 Deceleration (phase shift) of light by passing
through an object

http://www.microscopyu.com/tutorials/java/phasecontrast/phasespecimens/index.html
194

Köhler Illumination

… was established to guarantee optimal

illumination of objects.
This illumination is usually also a
prerequisite for different contrast methods
(phase contrast, differential interference
contrast) to work, as the necessary
components are optimized for Köhler
illumination.
In order to get a Köhler illumination, you
have to focus the object first, then you
close the field iris, so that just the middle
part of the view field is illuminated (if
necessary you have to center the light
path) and then the vertical position of the
condensor is adjusted so that the borders
of the field iris appear sharp and focussed.

195

97
 Correct and wrong illumination
field iris at wrong vertical position of the condensor

light path not centered Correct Koehler illumination

196

Specifications of objectives

• Magnification (10x, 20x, 40x…): total

magnification is given by objective
magnification and occular magnification (or
magnification of the lens in front of the CCD
camera)
• Immersion medium: air, oil – or water
High magnification (40x – 100x) with oil or
water
• additional features (e.g. suitability for
fluorescence due to low autofluorescence of
the glass: Neofluar..)
• contrast features: e.g. phase contrast (e.g.
Ph1)
• correction of lenses for chromatic
abberations (e.g. Apochromat)
• correction of lenses for planarity of focus
over the view field (Plan)

197

98
 Contrast enhancement in transmitted light
microscopy

1. Staining of structures
(e.g. nucleus: blue with
hematoxylin, antigen:
brown with immuno-
histochemistry)

2. Phase contrast:
Making use of the
phase of light when
it passes an object

3. Differential-Interference contrast
(Normarski): Making use of light
polarization and its change through
objects to generate a contrast 198

Phase contrast
Unstained objects such as cells
slow down the light (the phase of
the passing light) by ¼ λ. Phase
contrast rings in the objective can
accelerate the light, which does
not pass through cells by ¼ λ, the
resulting difference of ½ λ causes
an interference, which leads to
contrast enhancement.
¼λ

½λ

199
http://www.microscopyu.com/tutorials/java/phasecontrast/opticaltrain/index.html

99
 Phase contrast II

The phase contrast rings of the objektive and the condensor have to match each other in
diameter and have to be concentric. In addition the distance between them has to be
correct (which is the case at the Köhler illumination) – this is especially important for
higher magnification objectives. It is stated on the objektive which phase contrast ring has
to put in at the condensor (e.g. Ph1, Ph2...).

200

phase contrast III

wrong phase contrast ring correct Koehler illumination and
phase contrast

phase contrast rings not centered

201
http://www.microscopyu.com/tutorials/java/phasecontrast/microscopealignment/index.html

100
Illumination scheme of an inverted microscope

field iris

condensor with phase

contrast rings
Knob to adjust the
vertical position of the
condensor

screws for centering the

light path

202

Differential-Interference-Contrast (DIC)
(Normaski Contrast)
Before reaching the condensor, the light is polarized and
passes a double prism (Wollaston Prism), where it is split
into two beams with different directions and
perpendicular waves. These beams pass the sample,
where they are altered in intensity and phase etc. The
beams are focussed by the objective. In the focal plane
there is a second double prism, which combines the
beams again. After that, the beams are depolarized again.
Thereby the beams that have been altered differentially in
the sample can interfere with each other – and this
interference results in changes of the intensity and the
colour.
The outcome is a preudo-threedimensional image.

203

101
 Comparison

Phase Contrast – Differential Interference

HeLa cells – same view field

http://www.microscopyu.com/tutorials/java/phasedicmorph/index.html

204

Fluorescence Microscopy

Example: Triple-
Fluorescence-labeled
endothelial cells:
Red: Actin-Filaments
labeled with Phalloidin
Green: Membranes (DiO-C6)
Blue: Nuclei (DAPI-staining
of DNA)

205

102
 Basics of fluorescence microscopy
Fluorescent samples are
excited with light of an
appropriate wavelength
(through the objective), the
emitted fluorescence is
collected again by the
objective and is guided to a
dichroic mirror, which
separates the excitation light
from the emitted
fluorescence; the latter
passes an emission filter and
is detected (by eye or by
appropriate detectors such as
cameras)

Interactive Zeiss-Tutorials: http://zeiss-campus.magnet.fsu.edu/tutorials/ 206

Scheme of a fluorescence microscope

207

103
 Light sources for fluorescence excitation
1. Conventional light sources:
- mercury lamps:

- Xenon-lamps:

208

LED Light Sources

(light-emitting diodes: semiconductor devices)

from: http://zeiss-campus.magnet.fsu.edu/

104
 Metal Halide Lamps

from: http://zeiss-campus.magnet.fsu.edu/

Laser light sources

2. Laser (Light Amplification by
Stimulated Emission of Radiation):
Give just discrete wavelengths
(lines) – thus the choice of
excitation light is limited and
depends on the laser type.
Ar-laser: main lines at 488 nm and
514 nm, (and 458)
He/Neon: 543 nm, 633 nm
UV-Laser: 405 nm
Violet laser diodes: 405 – 420 nm
Advantages of laser light:
-high quality (parallel light beams)
-good for scanning
-high intensity
211

105
 Fluorescence Filter Cubes
The filter cube consists of:
1. Excitation filter: just the
correct excitation light
(wavelength) passes the filter
2. Dichroic mirror: is reflective
for the excitation light but
transmittent for the emission
light (the emitted fluorescence)
– separates excitation from sample
fluorescence light
3. Emission filter: filters the
emitted light so that just the
correct wavelength (e.g. in
double fluorescence) reaches
the detector
212

Characteristics of fluorescence filter sets

Excitation filter Emission filter

(Band Pass) (Band Pass)

dichroic
mirror
emission filter

213
excitation filter

106
 Example of a bandpass filter + nomenclature

214

Dualband filter sets: Simultaneous observation

of two different fluorophores (e.g. EGFP/DsRed)
dichroic mirror
Excitation Emission

215

107
 Monochromators as light source
A conventional light source (e.g. a Xenon lamp) is split by a
monochromator (e.g. a diffraction grid) to the spectral colours –
to produce light of a freely definable wavelength (320 – 700 nm).
This can be used instead of a fixed excitation filter.
One advantage is that this technology allows switching between
different excitation wavelengths within few milliseconds. This can
be important for excitation ratio imaging (e.g. Fura-2 Calcium
imaging etc.)
Polychrome V from
UV (320 nm) TILL Photonics

electronically
adjustable grid Red (700 nm)
216

Detection of the emitted fluorescence

- Visually via the occular of the microscope
- by a CCD camera (usually cooled to reduce the electronic noise).
The photons of the fluorescence hit a light sensitive chip (e.g. out
of 1300 x 1030 pixels), where electrons are released dependent
on the intensity of the fluorescence. Each chip can resolve a given
intensity range – e.g. 256 grey values for a 8-bit camera or 64000
grey values for a 16-bit camera.
The images can be shown on a computer monitor and saved on a
computer
- by photomultiplier tubes (PMT‘s): used often for scanning
devices such as confocal laser scanning microscopes. The gain
(voltage) of the PMT defines the sensitivity (electrons released for
a given number of photons that hit the detector). Often more
„noisy“ than CCD camera images. Averaging is used to smooth
the images (good images takes about 4 sec – while CCD require
just about 100 msec).
- old fashioned: film camera and sensitive film (e.g. 1600 ASA) 217

108
 Example for a fluorescence microscopy
experiment
- Cells transfected with fluorescent fusion proteins of a transcription
factor and its inhibitor (appear in the cytosol);
- addition of leptomycin B (LMB) to block nuclear export. This leads to
accumulation in the nucleus indicating continuous nucleo-cytoplasmic
shuttling

218

Example for a fluorescence microscopy

experiment II
Fluorescence was quantified in the nucleus and in the cytosol of the same cell
after different time points > shows the kinetics of nuclear accumulation by the
change of the cytosolic/nuclear ratio.
The data were fitted by nonlinear regression analysis (single exp. decay) –
leading to the half time of the nuclear import process.

5
cytosolic/nuclear fluor.

0
0 20 40 60 80
219
min

109
 Protocol of an immunofluorescence staining

• Fixation: 15 min 4% Paraformaldehyd

• 3x 5 min mit TBST wash (50mM Tris-
HCl pH7.4, 150 mM NaCl, 0.1%Triton)
• Block: 1 h at RT with 3% BSA in TBS
• Incubation with 1. Ab: anti-IκB (rabbit
polyclonal, sc-371 Santa Cruz)
1:300 in TBS/3% BSA, over night at
4°C (or 1 h at 37°C).
• 2x 5 min wash with TBST, 1x 5 min
with TBS
• Incubation with Alexa488 goat anti-
rabbit 1:2000 in TBS/BSA: 1 h at 37°C
• 3x 5 min wash with TBST, 1x 5 min
with TBS
• Mounting
220

Combinations of transmitted light and fluorescence

IP-Lab Software
A) direct ImageJ software
acquisition with
both light sources

B) Separate acquisition of fluorescence and phase contrast and merge

or blending (e.g. with ImageJ or other software)

221

110
 Interactive Microscopy Demonstrations
Very recommendable:
http://micro.magnet.fsu.edu/

1. Optical resolution:
http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html
http://www.microscopyu.com/tutorials/java/lightandcolor/refraction/index.html
2. Köhler Illumination:
http://www.microscopyu.com/tutorials/java/kohler/index.html
3. Phase shift of light by an object
http://www.microscopyu.com/tutorials/java/phasecontrast/phasespecimens/index.html
4. Phase contrast
http://www.microscopyu.com/tutorials/java/phasecontrast/opticaltrain/index.html
http://www.microscopyu.com/tutorials/java/phasecontrast/microscopealignment/index.html

5. Objectives with adjustable working distance

http://www.microscopyu.com/tutorials/java/aberrations/correctioncollar/index.html

222

Confocal Laser Scanning Microscopy (CLSM)

Problem in conventional microscopy: light, which comes from outside of the focal plane
(above or below) gets to the detector (or eye) and is registered as blur, which decreases the
quality of the image
Solution: A pinhole (iris) is placed into the light path at a position, where it can block out-of-
focus light. By that means an optical section is imaged (with variable thickness starting with
approx. 0.8 µm) depending on the diameter of the pinhole.
Usually high quality excitation light is needed for that (e.g. coherent laser light with parallel
light beams). The result is a very sharp image without any blur from out-of-focus light with a
slightly higher resolution than with conventional epifluorescence microscopy.
Photomultiplier
confocal pinhole

Laser

dichroic mirror
Scanner
Objective

z-Motor 223

111
 Confocal microscopy removes the blur
from thicker objects

http://zeiss-campus.magnet.fsu.edu/tutorials/opticalsectioning/confocalwidefield/index.html

z-stack
Optical sectioning and 3D-
projections
Acquisition of a „z-stack“ (image slices along the z-
axis) allows reconstruction of a 3D-projection, which
can be shown as animation

projection 3D rendering

225

112
 Spectral imaging
Resolving spectral information on a pixel-by-pixel basis
• „Emission finger printing“: emission scan of a
microscopy sample („lambda stack“ of images) at a
given excitation wavelength (e.g. with Zeiss LSM
META systems or with Leica confocal microscopes…)
• Alternative: Excitation scan (at a constant emission
wavelength; e.g. using a monochromator light
source)
Leica concept
• Combinations of excitation and emission finger
printing (e.g using filter wheels)
• Increases the number of markers to be measured in
parallel
• Can be used to discriminate fluorophores with
overlapping spectra
• Can be used to discriminate specific fluorescence
from autofluorescence
Zeiss META concept
226

Spectral Imaging Confocal Microscopy

(with Emission Curve Analysis)
Leica Confocal Microscope TCS SP2: Monochromator in front of the detector
AOBS: Acousto-Optical Beam Splitter (instead of dicroic mirror)
META System of Zeiss: 32 PMT-detectors every 10.7 nm
(400 – 720 nm): simultaneous wavelength analysis.

lambda-stack

Spectral curve of a
region of interest

227

113
Zimmermann et al. Sample with overlapping
(FEBS Letters 2003) fluorophores

1 2 3 1
Emission curves separated
4 5 6 into 8 channels (left) or
2
2 channels (right)
7 8

Equation matrix for the

channel signals based on
reference intensities in the
channels (GFPn and YFPn)
and the unknown
contributions of the
fluorophores

Unmixed fluorescence
(pseudo-coloured)

Spectral imaging example I: CFP, GFP and YFP

http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html
229

114
 Spectral imaging example II: strongly
overlapping dyes
SYTOX Green (nucleus), Alexa Fluor
488 conjugated to phalloidin
(filamentous actin network), and
Oregon Green 514 conjugated to goat
anti-mouse primary antibodies
(targeting mitochondria).

Invitrogen Spectra Viewer

http://www.invitrogen.com/site/us/en/
home/Products-and-
Services/Applications/Cell-
Analysis/Labeling-
Chemistry/Fluorescence-
SpectraViewer.html

230

Separation of specific fluorescence from auto-

fluorescence by spectral imaging

231

115
 Example for Emission Fingerprinting on a Zeiss
LSM510 META: Separation of GFP and YFP

Acquisition of a
reference lambda
stack for the first
fluorophore (GFP)

Obtain the spectral emission curve for the first fluorophore and
repeat the procedure for the second fluorophore
Intensity
250

YFP
200

GFP
150

100

50

0
500 510 520 530 540 550 560
Emission wavelength (nm)

116
 Unmixing of a mixed sample
(GFP-Actin and YFP-membranes)

Emission stack

Unmixed image

Unmixing of signals in pathology samples

(Shown with the Nuance™-Software from Cambridge Research & Instrumentation)

image with mixed signals for

different markers

autofluorescence

Brightfield display 235

117
 „Realtime“ confocal microscopy, Spinning disk
confocal microscopy (with Nipkow-disks)

gentle scanning (less bleaching

> good for sensitive life cells
Detection of the signal with a CCD-
camera

http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html 236

Companies for confocal microscopes

• Zeiss: http://www.zeiss.de

• Leica: http://www.leica.com
www.confocal-microscopy.com

• Nikon: http://www.instrumente.nikon.de/

• Olympus: http://www.olympus.de/microscopy/

237

118
 Multiphoton Laser Scanning-Microscopy

A quantum physical phenomenon is used: at very high light

densities (using pulsed lasers, about 900 nm infrared light)
packages of 2 or more photons occur (just in the focal plane !).
These have the same energy as single photons of higher energy
(shoerter wavelength, e.g. 450 nm). Thus these photon packages
can excite a fluorophore, which emits then at for instance 520 nm
(mitted wavelength is horter than the excitation light wavelength !).
An important advantage is that the 900 nm light has a mucher
deeper penetration into tissue (approx. 1 mm), while conventional
excitation can image just down to 0.25 mm. Another advantage is a
reduced overall bleaching effect, as excitation photon packages
occur just in the focal plane.

238

Multiphoton Laser Scanning-Mikroskopie II

conventional excitation
(1-Photon > cone of
ecitation light)
2-Photon excitation:
only a spot of
excitation

239

119
 Special Fluorescence Microscopy Techniques

1. FRAP: Fluorescence Recovery After Photobleaching

2. FLIP: Fluorescence Loss in Photobleaching
3. FRET: Fluorescence Resonance Energy Transfer
4. FLIM: Fluorescence Lifetime Imaging Microscopy
5. FISH: Fluorescence In Situ Hybridization

240

FRAP: Fluorescence Recovery After Photobleaching

FRAP at the membrane
Non linear regression analysis
y = span (1-e-kx) + bottom

An image is taken – then a region of the cell is bleached by high laser intensity, followed by
a time series of images after bleaching. Briefly after bleaching the region is significantly
darker and then the fluorescence intensity increases again (fluorescence reoovery) due to
diffusion of molecules into the bleached area. The kinetics of recovery depends on the
diffusion coefficience; the extent of recovery (the plateau to which the fluorescence
recovers) is a measure of the overall mobility (the fraction of mobile molecules versus
molecules immobilized, e.g. to the cytoskeleton)
241
FRAP in the cytosol:

120
 inverse FRAP with novel fluorescent
proteins

Protocol: FRAP analysis on Zeiss LSM510

• Capture an image of the whole cell before bleaching
• Define a bleaching / scan region (and maybe in addition another scan region that is not
bleached)
• Perform a time series with 1 scan prebleach, about 70 iterations of bleaching with 100%
laser power and then 50-100 scans of the bleach region (and also the non-bleached control
region if you specified one)- a good time resolution can only be obtained if just the small
bleach region (and maybe the control region) is scanned - and not the whole cell;
averaging of 2 or 4 scans reduces the electronic noise and leads to better quantifications.
• Capture an image of the whole cell after the FRAP time series (with the same conditions as
the prebleach image – for calculating the total loss of fluorescence.
• If you want to save disk space: extract the FRAP region and save just this region instead of
the whole image
• It is recommended to use the WCIF version of ImageJ for analysis: You can open the LSM-
files with the built-in feature (which also allows opening the time values of the image
series). Measure the mean fluorescence in a control region or for the whole cell for both the
prebleach and the postbleach images and calculate the loss of overall fluorescence due to
the bleaching in the region of interest (this is necessary for obtaining correct recovery
values for the bleach region).
• Import the FRAP-image sequence, define a measurement region and apply the „intensity
versus time plot“ plug-in – this will draw a graph of the FRAP curve; clicking the list button,
shows a list of the numerical values (the first 4 parameters are dimension and position of
the region, the rest are the fluorescence intensity values).
• Copy the fluorescence raw data from the list to the corresponding column of an Excel
template

243

121
 • Calculate the difference of mean fluorescence from the background and normalize
the fluorescence values to 100% for the initial fluorescence.

• Divide the percent values by the correction factor calculated from the total loss of
fluorescence (e.g. if total fluorescence decreased from 1 to 0.9 then divide the
mean fluorescence of the FRAP regions for each time value by 0.9 to compensate
for the loss in total fluorescence). A similar compensation can be obtained by
normalizing the FRAP fluorescence values to the control scan region that was not
bleached. This method also compensates more exactly for the bleaching effect in
the course of scanning of the time series (this scanning-dependent bleaching
effect is opposed to the recovery of fluorescence in the bleach region due to
diffusion of non-bleached molecules in the bleach region). This “dynamic
correction” gives a somewhat better estimation of the curve (and the kinetics of
the recovery) – but leads in principle to results that are very similar to the curve
obtained with the “constant correction factor” (by calculating the total loss in
fluorescence based on the intensities of the images that were captured before and
after the FRAP-time series)

• For non-linear regression analysis (curve fit of the data to a single exponential
association algorithm): Copy the data to a fitting program (such as GraphPad
Prism) and perform the fitting with a “bottom to span” algorithm:

y = span × (1 − e− kx ) + bottom
244

FLIP: Fluorescence Loss in Photobleaching

… to determine the dynamic shuttling of molecules between different
compartments of the cell

A certain compartment A (e.g. the cytosol) 120

is repetitively bleached by the laser – and 100

cytosol

the fluorescence decrease in a different nucleus

80
compartment B is monitored by time lapse
microscopy. Molecules that shuttle from B 60

to A are bleached in A > thus the 40

compartment B gets dimmer when there is
20
a dynamic distribution of molecules
between A and B. 0
0 2 4 6

122
 FLIP to determine a nuclear export signal and a
nucleolar localization signal
NFκB inducing kinase

truncated NIK without the export sequence:

nuclear FLIP
(bleach in nucleus outside nucleoli)

125
nuclear
nucleolar
100

75

50

25

0
0 100 200 300 400 500 600 700
sec

FCS: Fluorescence Correlation Spectroscopy

… to determine diffusion coefficients and interactions between molecules.
The sample is illuminated by the laser at a very small spot, the movements
of molecules in this spot (in and out) cause fluorescence fluctuations, which
are analyzed by correlation functions

123
 FRET: Fluorescence Resonance Energy Transfer
Microscopy
Energy can be transferred between two fluorophores when they
are very close to each other (closer than 10 nm) and when the
emission curve of one (the energy donor) overlaps with the
excitation curve of the other one (the acceptor). This transfer of
energy does not happen via photons (!) but by a dipole-interaction
(a quantum physical phenomenon discovered by Theodor Förster
in 1946). As a result the donor fluorophore fluorescence becomes
weaker and the acceptor fluorescence increases.
The FRET effect decreases with the 6th power of the distance; the
distance of half maximal energy transfer is called Förster-Distance
R0 (for CFP and YFP it is approximately 5 nm). As the effect is
usually not detectable anymore at a distance higher than 10 nm it
is ideally suited for monitoring macromolecular interactions
(protein-protein or protein-DNA). By that means, not only the
interaction by itself can be detected, but also the location of the
interaction and its dynamics.
248

Principle of Fluorescence Resonance Energy Transfer

no FRET FRET
Donor Acceptor
donor fluor.
(CFP)

acceptor fluor.
(YFP)

E = R06/(R06 + r6) und

nm R0 = [κ2 × J(λ) × n-4 × Q]1/6 × 970
excitation excitation
R0 … Förster-Distance
emission r ….. real distance
Donor κ ….. Orientation factor
Acceptor J(λ) … spectral overlap
n … refractive index
Q … Quantum yield of fluor.
249

124
 Appropriate Fluorophore Pairs for FRET
Fluorophore Pair Comments

CFP / YFP Good combination for normal FRET microscopy using Hg-
lamps as light source and special filters. CFP is poorly
excitated by Ar-lasers, but: good excitation by blue laser
diodes
BFP / GFP BFP has inferior fluorescence properties

GFP / DsRed-variants Original DsRed is just fluorescent as tetramer, shows

complex maturation characteristics with green fluorescent
intermediates and tends to aggregate; DsRed2 is a dimer.
The new monomeric DsRed works very fine with GFP.
GFP / YFP Are very difficult to separate with filters (but can be used in
FLIM and with spectral analysis)
GFP / Cy3 or Alexa 546 Antibodies can be directly labeled with Cy3 or equivalent
Alexa dye and give FRET with a GFP chimera to which they
bind.
FITC / TRITC Classical FRET pair for labeled proteins (e.g. antibodies)

Alexa 488 / Alexa 546 alternatives as labeling dyes (superior to FITC and TRITC)
(Cy3 / Cy5) 250

FRET can be used to monitor protein-DNA

interactions
spectra of donor and acceptor (GFP-NF-κB Spektral analysis of a mixture: Increase in
and Tet-labeled DNA, respectively) acceptor fluorescence indicates FRET
1.2 GFP-NFkB (Em)
Tet (Em)
1.0
Tet (Ex)
relative fluorescence

0.8

0.6

0.4

0.2

Spectral overlap
0.0
480 500 520 540 560 580 600
nm

Excitation of GFP-Emission (512 nm)

GFP (488 nm)

GFP Tet-Emission (540 nm)

GFP-NF-κB FRET
DNA Tet-label 251

125
 FRET can be applied to visualize the interaction
of signaling molecules in living cells

ECFP and EYFP-Scans decrease increase in

1,2 in Donor Acceptor
EYFP (Em) Emission Emission
1,0 EYFP (Ex)
relative fluorescence

ECFP (Em)
0,8 ECFP (Ex)

0,6

0,4
FRET

0,2
Spectral overlap
0,0 1 nm
350 400 450 500 550 600
nm
X Y
Förster Distanz R0 for ECFP and EYFP: ca. 5 nm (50%
FRET Effizienz): > no FRET-Signal beyond 10 nm. Donor Acceptor

252

Overview of FRET-Microscopy Techniques

1. Acquisition with FRET filter set (donor excitation and acceptor

emission): Problem: coexcitation of the acceptor at the donor
wavelength > false positives
2. Acquisition of a ratio image of acceptor fluorescence at donor
excitation and donor fluorescence at donor excitation
2-Filter FRET Microscopy: Just works when there is equal expression
of donor and acceptor (e.g. in fusion protein, biosensors)
3. 3-Filter FRET Microscopy
4. Determine the kinetics of donor fluorescence bleaching (this is
slower in the presence of a FRET acceptor, as part of the bleaching
energy is transfered to the acceptor)
5. Donor recovery after acceptor photobleaching
6. fluorescence lifetime microscopy– FLIM; fluorescence lifetime of the
donor decreases in the presence of a FRET acceptor
253

126
 Spectral crosstalk of donor and acceptor

ECFP and EYFP-Scans

1.2

1.0
raw FRET-channel:

relative fluorescence
0.8

0.6
Donor Excitation +
Acceptor Emission
0.4

0.2

0.0
340 360 380 400 420 440 460 480 500 520 540 560 580 600
nm

Excitation window Emission window

of donor of acceptor

Problems:
1. Co-excitation of the acceptor at the Donor-excitation wavelength >
Non-FRET-Fluorescence in the raw-FRET channel
2. Signal-overlap of donor into the acceptor channel > Non-FRET
fluorescence in the raw-FRET channel
254

2 Filter-FRET Microscopy (Ratio Imaging)

Ratio of donor emission and acceptor emission at the excitation
wavelength of the donor

ECFP and EYFP-Scans Limitations:

1.2 EYFP (Em) •concentration dependent
EYFP (Ex)
•donor and acceptor have to
1.0 ECFP (Em)
colocalize completely
relative fluorescence

ECFP (Ex)
0.8

0.6 just useful for FRET-

0.4 biosensors with covalent
0.2
linkage between donor and
acceptor
0.0
350 400 450 500 550 600 (equal-molar expression and
nm
100% colocalization)

excitation
emission-1 emission-2

image = Emission2 : Emission1 255

127
 3-Filter FRET Microscopy
3 Images are taken (under constant camera settings):
1. Donor (e.g. CFP-excitation and emission),
2. Acceptor (e.g.YFP-excitation and emission – this signal is not affected by FRET
3. FRET-Filter (raw FRET: CFP-excitation and YFP-emission).

A normalized FRET signal (image) can be calculated by using correction factors

obtained with single stained samples:

FRETc = IFRET - corrCFP x ICFP – corrYFP x IYFP

corrCFP : ca. 0.59

corrYFP : ca. 0.18

CFP / YFP neg. control CFP-YFP pos. control 256

FRET microscopy example

corrected FRET = IFRET - corrCFP x ICFP – corrYFP x IYFP
sample Donor Acceptor FRET corr. corrected
channel channel channel factor FRET

CFP alone 100 0 60 0.6 0

YFP alone 0 100 20 0.2 0

non-bound 100 100 80 0

CFP + YFP
bound CFP-YFP 100 100 160 80

Donor Acceptor corrected FRET normalized FRET

Normalized FRET
(normalized to diff.
neg. expression levels):
control

=
×

sample

257

128
 FRET Microscopy by analyzing the kinetics of
donor bleaching

… this is slowed down in presence of a FRET acceptor

time series of images time series of images

436 nm 476 nm 436 nm 476 nm

ECFP EYFP ECFP

FRET

CFP-Protein alone

CFP- and YFP-Protein

258

Donor-bleaching advantages:
kinetics concentration independent
donor and acceptor don‘t have to
colocalize completely
Limitation: requires external control,
difficult to obtain a FRET-image
Probe mit FRET
single exponential decay

y = A 0 .e −kt + offset
Probe ohne FRET
y... Fluor. Signal
A0... starting signal
k... decay constant
t... time
offset... final value

Fluorescence half time Tau: τ = 0.69/k

FRETeff. E = 1 - (τ without / τ with Akzeptor.)

259

129
 FRET Microscopy by acceptor bleaching and
monitoring donor recovery
(do not use for CFP / YFP)

Donor recovery after acceptor bleaching: Donor Acceptor

An image of the donor in the presence of FRET
the acceptor is taken, then the acceptor
is bleached (partially), followed by
acquisition of a second donor image

Donor Acceptor

260

Visualisation of biochemical reactions by FRET

microscopy (e.g. phosphorylations)
Detection of the auto-phosphorylation of EGF-receptor on Tyrosine residues using
GFP-EGFR fusion protein and Cy3-labeled anti-P-Tyr antibodies
(donor recovery after acceptor photobleaching technique)

erb2 -P
GFP
Cy3

GFP Ratio image

261

130
 FLIM: Fluorescence Lifetime Imaging Microscopy

The lifetime of donor fluorescence (usually in the nanosec. range) is

reduced in presence of a FRET acceptor.
This lifetime can be determined by a special variant of microscopy. Usually a
pulsed or a modulated laser is used for excitation. The fluorescence decay
(Time Domain) or the phase shift (Frequency Domain) of the emission
compared to the excitation is a measure of the fluorescence lifetime.

fluor. image FLIM image

262

FRET-Biosensors I

Caspase 3-Biosensor:
Apoptosis (Activation of caspase 3) is detected with a CFP-YFP fusion
protein in which CFP and YFP are separated by a caspase 3 cleavage site.
Without apoptosis: FRET, with apoptosis: no FRET

CFP YFP
Caspase 3

-…DEVD…- FRET

no FRET

263

131
 Other examples for FRET-Biosensors
Calcium-Biosensor: Ca2+-sensitive Calmodulin and a Ca2+/Calmodulin-binding
M13 domain are spliced between CFP and YFP (additional localization
sequences can be added – e.g. signal peptide and ER retention sequence). A
change in the calcium concentration leads to a change in the conformation of
the linker and thus to an alteration of the FRET signal.

Ca2+ YFP
CFP YFP FRET
CFP
Calmodulin M13
Ca2+

PKA Activity sensor: CFP and YFP separated by a PKA-substrate sequence

and a 14-3-3 domain, which binds phospho-serine of the PKA substrate
domain.
FRET
PKA-substrate 14-3-3
CFP YFP
PKA
CFP YFP
P

264

Digital Image Analyses

Often used: ImageJ (scientific Freeware: http://rsbweb.nih.gov/ij/ )

Different operations can be performed: contrast enhancement,

smoothing, background subtraction, measurement of fluorescence
intensities…
– and further more even math with images can be done (e.g.
dividing one image by another one; each pixel value is divided by
the corresponding pixel value of the second picture – at the same
pixel coordinates)

265

132
 Division of images
with ImageJ

266

PixFRET Plugin for ImageJ

133
 FRET analysis with self-written ImageJ macro

neg. control

14
12
10
8
6
4
2
0
Negative IKK1+Myc
sample IKK2+ Myc
1 sample 2
Control

sample

In Situ Hybridisation (ISH)

… for specific detection of DNA or mRNA sequences.

A labeled DNA- or RNA is hybridized to the target sequence in situ
(in the cell or the tissue) and detected

Applications:
1. Detection or (semi-) quantification of mRNA
2. Detection of DNA-sequences in chromosomes (e.g. translocations,
mutations, loss of genes…)

http://www.cytochemistry.net/In_situ.htm
http://osiris.rutgers.edu/~smm/in_situ_hybridization.htm

269

134
 Principle of ISH
- a labeled“ probe (e.g. DNA, labeled with Biotin-dUTP by Nick-Translation or an
oligonucleotide, labeled by terminal deoxynucleotidyl transferase, TdT) diffuses
into the cell and hybridizes with the target sequence. Addition of formamide to the
hybridization buffer lowers the specific hybridization temperature, so that at 37°C
only specific target sequences are bound. The probe is then detected via
fluorescence (fluorescence in situ hybr., FISH) or by enzyme activity (e.g. HRP
and colour reaction).

FITC

270

ISH: Advantages and Disadvantages of various probes

Probe type Advantages Disadvantages

DNA (double strand) Easy to use Reannealing during hybridization
Subcloning unnecessary (decreased probe availability)
Choice of labeling methods Probe denaturation required,
High specific activity increasing probe length and
Possibility of signal amplification decreasing tissue penetration
(networking) Hybrids less stable than RNA probes
DNA (single strand) No probe denaturation needed Technically complex
No reannealing during hybridization Subcloning required
(single strand) Hybrids less stable than RNA probes
RNA Stable hybrids (RNA-RNA) Subcloning needed
High specific activity Less tissue penetration
No probe denaturation needed
No reannealing
Unhybridized probe enzymatically
destroyed, sparing hybrid
Oligonucleotide No cloning or molecular biology Limited labeling methods
expertise required Lower specific activity, so less
Stable sensitive
Good tissue penetration (small size) Dependent on published sequences
Constructed according to recipe Less stable hybrids
from amino acid data Access to DNA synthesizer needed
No self-hybridization
from: Feldman, RS, Meyer, JS, and Quenzer, LF (1997). Principles of Neuropsychopharmacology. Sunderland, MA: Sinauer Associates, Inc. Pages 31-35.

more recently: BAC probes (can be obtained from collections): high sensitivity for single copy genes… 271

135
 Examples for ISH
FISH with interphase nuclei

Detection of a target-mRNA in cryo-

Fluorescence In Situ-Hybridization on
Metaphase-chromosomes
or paraffin sections

Detection with 33P

Detection with alkal. Phosphatase

272

mRNA-FISH Protocol
• Cells are fixed with freshly made 4% formaldehyde in PBS, pH 7.4 for 15 min at
room temperature. All solutions should be made in Molecular Biology grade
ultrapure water (no RNase). Wear gloves at all times and use sterile disposable
pipets and tips.
• After rinsing in PBS (3 X 10 min. each), cells are permeabilized with 0.5% Triton
X-100 in 1X PBS for 10 min at 4oC.
• Cells are then rinsed in PBS (3 X 10 min. each) and then in 2X SSC (1 X 5 min.).
• 100 ng of nick translated probe (containing digoxigenin dUTP) and 20 ug of
competitor E. coli tRNA per coverslip are dried down in a Speed Vac (Savant).
This is a good starting place but you may have to titrate your specific probe.
• 10 μl of deionized formamide is added to the dried DNA.
• The probe and tRNA are denatured by heating for 10 min at 90oC. The probe is
chilled on ice immediately.
• 10 μl of Hybridization buffer (20% dextran sulfate + 4X SSC) is added to the
denatured probe so that the final concentrations in the hybridization mixture
are 5 ng/ml of probe, 1 ug/ml of E. coli tRNA, 2X SSC, and 10% dextran sulfate.
• 20 μl of hybridization mixture/probe is placed onto each coverslip.
• Coverslips are inverted onto a slide and sealed with rubber cement and
incubated in a humid chamber for 16 hrs. at 37oC.
• After rinsing in 2X SSC/50% formamide at 37oC, 2X SSC and 1X SSC at room
temperature for 30 min. each, the coverslip containing cells are incubated in 4X
SSC/0.25% BSA/2ug/ml anti-digoxigenin antibody for 60 min. in a humid
chamber at room temperature in the dark.
• Coverslips are then rinsed in 4X SSC (1 X 15 min.) at room temperature, 4X
SSC/0.1% Triton X-100 (1 X 15 min.), and 4X SSC (3 X 10 min. each).
• Coverslips are mounted in fluorescence mounting medium.
Modified from: Jiménez-García, L. and D.L. Spector. 1993. Cell 73, 47-59.
273

136
 Superresolution Microscopy I
STED: Stimulated Emission Depletion

A second laser (depletion laser) „trims“ the excitation spot (point-spread function, PSF) to a
smaller size. Resolution appr. 80 nm.

274
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html

Superresolution Microscopy II
Structured Illumination Microscopy (SIM)

A second laser (depletion laser) „trims“ the excitation spot (point-spread function, PSF) to a
smaller size. Resolution appr. 80 - 100 nm.

normal image SIM image

A known pattern is projected into the

image plane at different angles and
interferes with sample structures,
creating Moiré pattern. Superresolution
information can now be captured by the
microscope from these structures by
mathematical algorithms.
275
(from www.zeiss.de)

137
 Superresolution Microscopy
- by single molecule detection
STORM: Stochastic Optical Reconstruction Microscopy using single fluorescent molecules
PALM: Photoactivated Localization Microscopy
Resolution: appr. 30 nm, based on statistical calculation of the center of a Gaussian Fit of a
single molecule. Requires a sensitive camera (e.g. EMCCD: Electron-multiplying charge-
coupled device cameras) – and some software, but no specific hardware

276
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html

Research Methods - Overview

• cell culture systems

• labelling and transfection of cells
• analyses of cellular components
• analyses of molecular interactions
• fluorescence measurements
• microscopy
• flow analysis (FACS)
• analyses of cellular processes (proliferation, apoptosis..)

277

138
 Flow Cytometry

Some contents are inspired by “Fluorescence Spectroscopy in

Biological Research”
by Robert F. Murphy

Definitions:

• Flow Cytometry
– Measuring properties of cells in flow
• Flow Sorting
– Sorting (separating) cells based on properties measured in
flow
– Also called Fluorescence-Activated Cell Sorting (FACS)

278

Basics of Flow Cytometry

•Cells in suspension

Fluidics •flow in single-file

through
•an illuminated volume
where they
•scatter light and emit
Optics fluorescence
•that is collected,
filtered and
- Flow Cytometry, Flow Analysis).
•converted to digital
Electronics values - Flow Sorting, Fluorescence
Activated Cell Sorting, FACS
•that are stored on a
279
computer

139
http://probes.invitrogen.com/resources/education/

280

Fluidics

• Need to have cells in suspension flow in single

file through an illuminated volume

• In most instruments, accomplished by

injecting sample into a sheath fluid as it
passes through a small (50-300 µm) orifice

281

140
 Fluidics II
• When conditions are right, sample fluid flows in a central core
that does not mix with the sheath fluid
• This is termed Laminar flow

• Whether flow will be laminar can be determined from

the Reynolds number
V is the mean fluid velocity in (SI units: m/s)
D is the diameter (m)
μ is the dynamic viscosity of the fluid (Pa·s or
N·s/m²)

When Re < 2300, flow is always ν is the kinematic viscosity (ν = μ / ρ) (m²/s)

laminar ρ is the density of the fluid (kg/m³)
When Re > 2300, flow can be Q is the volumetric flow rate (m³/s)
turbulent A is the pipe cross-sectional area (m²)
282

Flow Cell

Injector
Tip Sheath
fluid

Hydrodynamic
Fluorescence Focusing
signals

Focused laser
beam

283
Purdue University Cytometry Laboratories

141
 Hydrodynamic Focusing

The figure shows the

mapping between the
flow lines outside and
inside of a narrow
tube as fluid
undergoes laminar
flow (from left to
right). The fluid
passing through cross
section A outside the
tube is focused to
cross section a inside.
284
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

Hydrodynamic Focusing II

Example: Focusing of ink by

laminar flow into a capillary

Notice how the ink is focused

into a tight stream as it is drawn
into the tube under laminar
flow conditions.

In flow cytometry:
Sample container and the sheath fluid container are put under defined air
pressure. Laminar flow is maintained (and hydrodynamic focussing is
achieved) by a precise control of the pressure difference between the sample
container, the sheath fluid container and the atmosphere

285
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

142
 Flow Chamber

Different types are possible,

a commonly used type:

Flow
through
cuvette
(sense in
quartz)

286
H.B. Steen - MLM Chapt. 2

Optics: General scheme

PMT
4

Dichroic PMT
Filters 3
Flow cell
PMT
2

Bandpass Filters

PMT
1

FSC Laser

SSC

287
original from Purdue University Cytometry Laboratories;
modified by R.F. Murphy and J. Schmid

143
 Forward Scatter (FSC)

When a laser light source is

used, the amount of light
scattered in the forward
direction (along the same axis
that the laser light is traveling)
is detected in the forward
scatter channel
The intensity of forward
scatter is mainly proportional
to the size and surface
properties of cells (or other
particles)

288

Forward Scatter
Detector

Usually not a sensitive PMT detector

(because the light intensity is high)
– but rather a photodiode detector,
which can be set in log10-
increments (E-1, E0, E1...), and a
linearity gain factor (1.0 – 9.99).
A blocking bar prevents the direct
laser light from hitting the detector.
A predefined FSC-Wert is often used
as threshold to discriminate
between cells and dust particles

289

144
 Forward scatter threshold to ignore debris and
small particles or cells

290

Side Scatter
• When a laser light source is (SSC)
used, the amount of light
scattered to the side
(perpendicular to the axis that
the laser light is traveling) is
detected in the side or 90o
scatter channel
• The intensity of side scatter is
mainly dependent on
subcellular structures (e.g.
granules, vesicles…)

291

145
 90 Degree Light Scatter (SSC)

Laser

FSC Detector

90° scattered light

(side scatter) is
detected with a
photomultiplier
detector, where the
sensitivity can be set
in Volt. Amplification
scale can be linear or
logarithmic.
SSC detector

292
Purdue University Cytometry Laboratories

Optics: Fluorescence Channels

dichroic mirrors

bandpass filters

146
 Common 350 457 488 514 610 632
Laser 300 nm 400 nm 500 nm 600 nm 700 nm Fluorophores
Lines PE-TR Conj.

Texas Red

PI

Ethidium

PE

FITC

cis-Parinaric acid

294
Purdue University Cytometry Laboratories

Sorting of cells
FL1

488 nm Laser
FSC
Sensor

Fluorescence
-
FSC

charged - + Gating
control unit
electrodes + -

Sorted
cells

295
Purdue University Cytometry Laboratories; modified

147
 296

Sorting Mode
If there are 2 or more cells in one droplet, Target-Cell
and the droplet contains target and non
target cells, then there is a „sorting conflict“,
which has to be solved by defining an
appropriate sorting mode:
Non-target cell

• Exclusion-Mode: droplets containing non target cells are dismissed

even if they contain target cells > high purity, maybe lower yield
• Recovery-Mode: droplets containing target cells are collected, even if
they also contain non-target cells > high yield, maybe low purity.
• Single Cell Mode: only droplets with single target cells are collected
> high purity, maybe low yield, high counting accuracy.

297

148
 Overlap of fluorescence signals and
„Compensation“

298

When cells are labeled with 2 fluorophores (e.g. FITC and

„Compensation“ PE), there might be a signal crosstalk between the detection
channels (dependent on detector voltage settings). This can
be compensated in the follwing way: measuring a sample
containing only fluorophore 1: if there is crosstalk, you see
an elevated intensity in both channels – using the
compensation control of the software, you can bring the
fluorescence signal in the wrong channel down to the
background fluorescence. With a sample stained only with
fluorophore 2 you do the same for the other channel. This
compensation is usually just valid for the detector settings
at which it was set.

compensation for fluorophore 2 299

compensation for fluorophore 1

149
 Compensation II

before after
compensation compensation

> true double positives can

be determined

300

FACS-Graphs counts

Histograms: the intensity of one

channel is divided into classes
(often 1024, x-axis) and the
frequency of „events“ (cells)
scored into these 1024 classes. 0

FL1
Dot Plots: 2 different parameters
are plotted on the x- and y-axis;
each cell is a spot on this graph
according to its parameter
intensities.
FL2

Correlation between
histograms and
dotplots
301

150
 FACS-Graphs II
FL1

Density Plots: The frequency of events

(cells) in various areas of a dot plot is
colour coded to highlight the peaks
FL2

Contour Plots: The frequency of events FL1

(cells) in various areas of a 2D-plot is

visualized by lines representing equal
probability (similar to contour lines of
mountains on a map)
FL2

3D-Plots: The frequency of events

measured for 2 parameters is illustrated
by a third axis (z-axis) in a 3D manner.
FL2
302

Statistics and „Gating“

Region R1: 69.3%
Regions: can be defined in (Polygon-Region)
different forms (polygons,
ellipses..). These can be used for
statistics of cells falling into a
certain region – but also for
„gating“ that means rejecting
certain cells inside (or outside) a
certain region for analysis (or Marker M1: 67.5%
data acquisition) – or for instance
for sorting of cells.

Marker: Upper and lower limits in

histograms for quantification
UL (upper left): 0.06%
Quadrants: split the 2D graph in 4 UR (upper right): 29. 88%
regions, by the coordinates of the LL (lower left): 0.64%
point where 2 axis intersect. LR (lower right): 69.44%
Allows fast and simple statistics.

303

151
 Research Methods - Overview

• cell culture systems

• labelling and transfection of cells
• analyses of cellular components
• analyses of molecular interactions
• fluorescence measurements
• microscopy
• flow analysis (FACS)
• analyses of cellular processes (proliferation, apoptosis..)

304

Analytical Applications of Flow Analysis

• Leukocyte analyses
• Phenotyping of cells (CD-Marker)
• Immunofluorescence stainings
• cell cycle analyses (DNA-content)
• Chromosome analyses
• Proliferation assays (Brd-U incorporation)
• Apopotosis assays (Annexin V, TdT, JC1)
• Calcium flux-measurements
• etc.

305

152
 Leukocyte analyses Phenotyping of cells

CD8-positive T-cells

CD4-positive T-cells
306

cell cycle analyses by staining of DNA with PI

diploid

G2M
Chromosome content G0
G0/G1 G1
doubled diploid s
G0G1
G2/M
apoptotic
cells
S
s G2 M
0 200 400 600 800 1000
2N 4N
DNA Gehalt
Propidium-Iodide Fluorescence of
permeabilized cells after digestion of RNA: 307
fluor. depends on DNA content

153
 Protocol of a Propidium-Iodide Staining
Adherent cells:
trypsinized, suspended in medium + 10% FCS, centrifuged (1000 rpm, 5 min), Pellet
suspended in PBS (1 ml)
Suspension cells:
Centrifuged (1000 rpm, 5 min), Pellet suspended in PBS (1 ml)
Fixation with EtOH:
Pipet cell suspension into 2.5 ml absolute EtOH (final concentration approx. 70%) - or vortex
the suspension at half speed while adding the EtOH) – to prevent clustering of cells during the
fixation. Incubate on ice for 15 min (or over night at –20°C).
Alternative fixation with paraformaldehyde:
Pipet the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r.t.
Staining:
Pellet the cells at 1500 rpm for 5 min, Suspend the pellet in 500 µl PI-solution in PBS:
50 µg/ml PI from 50x stock solution (2.5 mg/ml), 0.1 mg/ml RNase A, 0.05% Tritin X-100
Incubate for 40 min at 37°C
Add 3 ml of PBS, pellet the cells (1500 rpm, 5 min) and take off the supernatant
Suspend the pellet in 500 µl PBS for flow analysis
(you can also leave about 500 µl of the diluted staining solution on the pellet and suspend the
cells in this solution > less loss of cells when you take off the sup.) – the rest of the staining
solution does not interfere with the flow analysis.
Flow analysis:
Approximate settings (on FACSort):
FL1: 570 V log. (e.g. if you want to detect GFP) 308
FL2: 470 V linear

Chromosome Analyses

309

154
 Analysis of Proliferation
(BrdU-labeling of S-Phase Cells)

Cells are cultured for a given

time in medium containing
FITC-anti- Bromodeoxy-Uridin (BrdU). This
BrdU Fluor. nucleotide analogon incororates
into newly synthesized DNA (of
cells in S-phase) – and can be
detected with anti-BrdU
antibodies (e.g. FITC labeled).

Propidiumjodid Fluor. (PI)

310

Staining of apoptotic cells with JC-1

JC-1 (5, 5´, 6, 6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazol-carbocyanine iodide)
is a dye, which incorporates in to mitochondrial membranes, where the fluorescence
depends on the membrane potential. In normal cells (with intact mitochondrial
membrane potential) it builds aggregates and emits mainly red fluorescence, in
apoptotic cells (where the membrane potential breaks down) it occurs in monomers
and emits mainly green fluorescence. This can be detected by flow analysis or
microscopy.

311

155
 Protocol of a JC-1 Staining of Apoptotic Cells
JC-1 is prepared as a 1000x stock solution in DMSO (5 mg/ml).
For the staining of adherent cells it is diluted in medium to 5 µg/ml (with
vortexing during the dilution to prevent the formation of precipitates); the JC-
1 containing medium is added to the cells, followed by incubation for 10 min
at 37°C (or RT for 15 min).
Subsequently the cells are washed twice with PBS, trypsinized, suspended in
500 µl PBS and analyzed by flow analysis.
Suspension cells (lymphocytes): suspend 1:1 with 10 µg/ml JC-1 in medium
(final conc.: 5 µg/ml)

Approximate detection settings on FACSort:

FL1: 360 V (log)

FL2: 310 V (log)
Compensation : FL1-7% FL2 und FL2-74% FL1 312

Detection of apoptotic cells by PI-staining of

permeabilized cells (cell cycle analysis)

Fragmented DNA emits

lower fluorescence then
cells with the normal
diploid DNA content – this
„Sub-G0/G1“ population
reflects apoptotic cells
with fragmented DNA )at
a late stage of apoptosis)

313

156
 Detection of apoptotic cells with Annexin V

Fluorescence-labeled
Annexin V: binds to
phosphatidylserine, which
is normally on the inner
leaflet of the membrane,
but which is flipped to the
outside during apoptosis

Discrimination between necrotic and

apoptotic cells
PI-pos/Annexin-neg.: necrotic
PI-neg/Annexin-pos.: early apoptotic
PI-pos/Annexin-pos.: late apoptotic, or
necrotic with large holes in the membrane
(where Annexin V can get through)
314

Calcium Flux Determination by Flow

Analysis

Cells are stained with a calcium sensitive fluorophore (INDO-1 or

better Fluo4); after a stimulus, the kinetics of the fluorescence signal
is measured (monitoring calcium influx).

315

157
 Sources

• Purdue Univ. Cytometry Laboratories

http://www.cyto.purdue.edu/
• Research Institute of Scripps Clinic: http://facs.scripps.edu/
• Invitrogen Tutorials
(http://www.invitrogen.com/site/us/en/home/support/Tutorials.html)
• BD Tutorials: http://www.bdbiosciences.com/support/training/itf_launch.jsp
• J. Paul Robinson (Purdue Univ.)
• Robert F. Murphy (Carnegie Mellon Univ., Pittsburgh)
• Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T.
Lindmo, M.L. Mendelsohn, eds.), Wiley-Liss, New York, 1990 –
abgekürzt: MLM
• Flow Cytometry: Instrumentation and Data Analysis
(M.A. Van Dilla, P.N. Dean, O.D. Laerum, M.R. Melamed, eds.),
Academic Press, London, 1985 – abgekürzt: VDLM

316

Companies offering flow analysis equipment

• Becton-Dickinson
http://www.bd.com
• Beckman-Coulter
http://www.beckmancoulter.com
• Millipore:
http://www.millipore.com/
• Accuri (> now part of BD)
http://www.accuricytometers.com/
• Partec: http://www.partec.com/

317

158
 Free Software: Flowing Software 2
http://www.flowingsoftware.com

318

Flowing Software 2

Quadrants

Regions in histograms

319

159
 320

Cell cycle analysis with Flowing Software

• Define 3 histogram regions (H1, H2, H3:
G0/G1, S and G2/M-phase, respectively)
• Activate the region control tool: Cell Cycle
• Define the G2 peak multiplier and peak width
(right click in the cell cycle window)
• Choose active control (after right clicking)
• Create statistics (by right clicking into the
histogram window)
• Create a Stat.List by right-clicking into the
Statistics window
• Ctrl-N loads the next file, adjusts the H1-H3
regions automatically and calculates the cell
cycle phases (H1-H3)

321

160
 Tissue Cytometry: Quantitative Image Analysis of
microscopy samples
Single-cell recognition (e.g. based on DAPI-nuclear fluorescence) > generation of cell masks for
quantification of signals (e.g. in different fluorescence channels) > scattergrams can be derived similar to
flow cytometry (for the cells in their original tissue environment!) > Gating and statistics are possible

http://www.tissuegnostics.com
Similar evaluations can be done with
ImageJ using automatic threshold and the
„Analyze particles“ feature.

Tissue Arrays for quantitative comparison of samples

> equal staining conditions for all samples

CA PIN
Normal

Pat. #1
Pat. #2
Pat. #3
Pat. #4
Control
Gingiva
Cystectomy

Example of a
prostate tissue
array

161
 Multi-parallel coordinate plots for visualization
of several parameters in parallel
Can be done with Freeware (Mondrian:
http://www.theusrus.de/Mondrian/index.html)
One event (e.g. one cell) is represented by a line linking
several y-axis (for the different parameters e.g.
fluorescence signals); a „population“ can be selected
and is highlighted also for the other parameters. The
data density can be reduced (using a so called alpha-
factor) to obtain better visibility of numerous data
points.

1 2 3 4 5 6 7 8 9 10

The density of lines (events) can be visualized in colour-

coded manner (example: 10-parameter parallel
coordinate plot generated with MatLab) > fast intuitive
visualization of multiparameter data sets

162