Notes

The rapid breeding cycle of mice makes them particularly well suited for the
production of inbred strains in which the heterozygosity of alleles that is normally
found in randomly outbred mice is replaced by homozygosity at all loci. Repeated
inbreeding for 20 generations yields an inbred strain whose progeny are
homozygous and identical (syngeneic) at more than 99% of all loci.
Recombinant inbred strains of mice are those in which two inbred strains (e.g.,
strains A and B) have been mated, resulting in a recombination within an interesting
locus (e.g., within the MHC locus). Subsequent inbreeding of the animals with this
recombination results in an inbred strain of mice bearing part of its MHC from strain
A and the other from MHC B. Animals from this strain can then be used to determine
which sub-regions of a locus contribute to which properties in the immune system of
the animal. Such strains were used to delineate the functions of class 1 versus class
2 proteins encoded by the MHC locus.
Two strains of mice are congenic if they are genetically identical except at a single
genetic locus or region. Any phenotypic differences that can be detected between
congenic strains must therefore be encoded in the genetic region that differs
between the two strains.
Immunoglobulin superfamily.
Class I Molecules Have a Glycoprotein Heavy Chain and a Small Protein Light Chain:
2-microglobulin does not contain a transmembrane region and is non-covalently
bound to the MHC class I chain. Sequence data reveal strong homology between the
3 domain of MHC class I, 2-microglobulin, and the constant-region domains found
in immunoglobulins.
Class II Molecules Have Two Non-Identical Glycoprotein Chains
Peptide binding by class I and II molecules does not exhibit the fine specificity
characteristic of antigen binding by antibodies and T-cell receptors. Instead, a given
MHC molecule can bind numerous different peptides, and some peptides can bind
to several different MHC molecules.

Although B- and T-cell receptor diversity is generated through genomic

rearrangement and gene editing, MHC molecules have opted for a combination of
peptide binding promiscuity and the expression of several different MHC molecules
on every cell. Using this clever combined strategy, the immune system has evolved
a way of maximizing the chances that many different regions, or epitopes, of an
antigen will be recognized.

The genes that reside within the MHC region are highly polymorphic; that is, many
alternative
forms
of
each
gene,

or alleles, exist within the population. The individual genes of the MHC loci (class I,
II, and III) lie so close together that their inheritance is linked. Crossover, or
recombination between genes, is more likely when genes are far apart. For instance,
the recombination frequency within the H-2 complex (i.e., the frequency of
chromosome crossover events during meiosis, indicative of the distance between
given genes) is only 0.5%. Thus, crossover occurs only once in every 200 meiotic
cycles. For this reason, most individuals inherit all the alleles encoded by these
genes as a set (known as linkage disequilibrium). This set of linked alleles is referred
to as a haplotype. An individual inherits one haplotype from the mother and one
haplotype from the father, or two sets of alleles.
In outbred populations, such as humans, the offspring are generally heterozygous at
the
MHC
locus,
with
different
alleles contributed by each of the parents. If, however, mice are inbred, each H-2
locus becomes homozygous because the maternal and paternal haplotypes are
identical, and all offspring begin to express identical MHC molecules. Certain mouse
strains have been intentionally inbred in this manner and are employed as
prototype strains. The MHC haplotype expressed by each of these strains is
designated by an arbitrary italic superscript (e.g., H-2a, H-2b). These designations
refer to the entire set of inherited H-2 alleles within a strain without having to list
the specific allele at each locus individually. Different inbred strains may share the
same set of alleles, or MHC haplotype, with another strain (i.e., CBA, AKR, and C3H)
but will differ in genes outside the H-2 complex.
The genes within the MHC locus exhibit a codominant form of expression, meaning
that both maternal and paternal gene products (from both haplotypes) are
expressed at the same time and in the same cells. Therefore, if two mice from
inbred strains possessing different MHC haplotypes are mated, the F 1 generation

inherits both parental haplotypes and will express all these MHC alleles.

As the sheath fluid moves, it creates a massive drag effect on the narrowing central
chamber. This alters the velocity of the central fluid whose flow front becomes
parabolic with greatest velocity at its center and zero velocity at the wall. The effect
creates a single file of particles and is called hydrodynamic focusing. Under optimal
conditions (laminar flow) the fluid in the central chamber will not mix with the
sheath fluid.
Light that is scattered in the forward direction, typically up to 20o offset from
the laser beams axis, is collected by a lens known as the forward scatter
channel (FSC). The FSC intensity roughly equates to the particles size and can
also be used to distinguish between cellular debris and living cells.
Light measured approximately at a 90o angle to the excitation line is called
side scatter. The side scatter channel (SSC) provides information about the
granular content within a particle.
There
are
types. Long pass filters
light above a cut-off
short pass permit light
wavelength and band
light within a specified
wavelengths (termed a
All these filters block
absorption.

three

major

filter
allow
through
wavelength,
below a cut-off
pass
transmit
narrow range of
band
width).
light
by

When a filter is placed

at a 45o angle to
the oncoming light it
becomes
a
dichroic filter/mirror. As
the
name
suggests, this type of
filter
performs
two
functions, first, to pass specified wavelengths in the forward direction and,
second, to deflect blocked light at a 90o angle. To detect multiple signals
simultaneously, the precise choice and order of optical filters will be an
important consideration.
Log amplification is normally used for fluorescence studies because it expands
weak signals and compresses strong signals, resulting in a distribution that is
easy to display on a histogram. Linear scaling is preferable where there is not
such a broad range of signals e.g. in DNA analysis.

Electrostatic
charging actually occurs at a precise moment called the break-off point, which
describes the instant the droplet containing the particle of interest separates from
the stream.
The difference between Eexcitation and Eemission is called Stokes Shift and this
wavelength value essentially determines how good a fluorochrome is for
fluorescence studies. After all, it is imperative that the light produced by emission
can be distinguished from the light used for excitation. This difference is easier to
detect when fluorescent molecules have a large Stokes Shift.
Fluorescent probes are useful in a wide range of applications including: identifying
and quantifying distinct populations of cells, cell surface receptors or intracellular
organelles; cell sorting; immunophenotyping; calcium influx experiments;
determining nucleic acid content; measuring enzyme activity, and for apoptosis
studies. By changing the excitation light and using more than one fluorochrome, it is
possible to analyze several parameters of the sample at any one time. This forms
the basis of multicolor fluorescence studies.
In a tandem dye, a small fluorochrome takes a piggy-back ride on another larger
fluorochrome. When the first dye is excited and reaches its maximal singlet state,
all its energy transfers to the second dye (an acceptor molecule), located in close
proximity. This activates the second fluorochrome, which then produces the
fluorescence emission. The process is called FRET (fluorescence resonance energy
transfer). It is a clever way to achieve higher Stokes Shifts and, therefore, increase
the number of colors that can be analyzed from a single laser wavelength.
An important principle of flow cytometry data analysis is to selectively visualize the
cells of interest while eliminating results from unwanted particles e.g. dead cells
and debris. This procedure is called gating.

A single-parameter histogram
Cells with the desired characteristics are known as the positive dataset. Ideally, flow
cytometry will produce a single distinct peak that can be interpreted as the positive
dataset.
However,
in
many
situations,
flow
analysis
is performed on a mixed population of cells resulting in several peaks on the
histogram. In order to identify the positive dataset, flow cytometry should be
repeated in the presence of an appropriate negative isotype control.
Isotype controls are primary antibodies that lack specificity to the target, but match
the class and type of the primary antibody used in the application. Isotype
controls are used as negative controls to help differentiate non-specific background
signal from specific antibody signal.
Isotype controls are a type of negative control designed to measure the level of nonspecific background signal caused by primary antibodies, based upon the tissue
type of the sample. Usually, the background signal is the result of immunoglobulins
binding non-specifically to Fc receptors present on the cell surface. For example,
antibodies raised in mice, particularly those of the IgG2a isotype, will bind strongly
to some human leukocytes regardless of the test antibody specificity. In this case
you would need a mouse IgG2a isotype control for use with human cells or tissues.
Isotype controls are most commonly used in flow cytometry experiments, but may
also be used in immunohistochemistry.
Selecting an Isotype Control Typically, an isotype control is matched to the host
species and isotype of your specific primary antibody. Sometimes, it is an antibody
that has been raised against an antigen that is not normally expressed in the target
tissue, e.g. DNP, but this is not always the case. When using directly labeled
primary antibodies, it is also necessary to make sure that the isotype control is
conjugated to the same fluorochrome or label as the test antibody. It is not sufficient
to use one with a spectrally similar fluorochrome.
Lymphocytes were stained with anti-CD3 in the FITC channel (x-axis) and anti-HLADR in the PE channel (y-axis). CD3 and HLA-DR are markers for T cells and B cells,
respectively.
The top right quadrant contains a few activated T cells (about 4% in this sample)
that possess some HLA-DR expression also.
Staining intracellular antigens like cytokines can be difficult because antibody-based
probes cannot pass sufficiently through the plasma membrane into the interior of
the
cell.
To
improve
the
situation,
cells
should
first be fixed in suspension and then permeabilized before adding the fluorochrome.
This allows probes to access intracellular structures while leaving the morphological
scatter characteristics of the cells intact.
The thymus organ supports the development of T cells and is located in the thorax.
Here, we report the existence of a second thymus in the mouse neck, which

develops after birth and grows to the size of a small lymph node. The cervical
thymus had a typical medulla-cortex structure, was found to support T cell
development, and could correct T cell deficiency in athymic nude mice upon
transplantation. The identification of a regular second thymus in the mouse may
provide evolutionary links to thymus organogenesis in other vertebrates and
suggests a need to reconsider the effect of thoracic thymectomy on de novo T cell
production.
We recognized that some of these cervical lymphoid organs displayed hallmarks of
thymus structure, including a medulla-cortex architecture marked by cytokeratin
expression, CD4+CD8+ double-positive (DP) and CD4+CD8 and CD4CD8+ singlepositive (SP) thymocytes with low and high levels of T cell receptor (TCR)/CD3
expression, respectively.
Fused cells are incubated in HAT medium (hypoxanthine-aminopterin-thymidine
medium) for roughly 10 to 14 days. Aminopterin blocks the pathway that allows for
nucleotide synthesis. Hence, unfused myeloma cells die, as they cannot produce
nucleotides by the de novo or salvage pathways because they lack HGPRT. Removal
of the unfused myeloma cells is necessary because they have the potential to
outgrow other cells, especially weakly established hybridomas. Unfused B cells die
as they have a short life span. In this way, only the B cell-myeloma hybrids survive,
since the HGPRT gene coming from the B cells is functional. These cells produce
antibodies (a property of B cells) and are immortal (a property of myeloma cells).
The incubated medium is then diluted into multi-well plates to such an extent that
each well contains only one cell. Since the antibodies in a well are produced by the
same B cell, they will be directed towards the same epitope, and are thus
monoclonal
antibodies.

B cell activation requires both

antigen engagement by the B-cell receptor (BCR) and direct contact with an
activated
CD4
TH
cell.
Both
events
are
facilitated
by
the
anatomy of the lymph node.