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Myelin Removal Beads

human, mouse, rat

Order no. 130-094-544

Contents leading to a higher purity and recovery of target cells, and thereby
highly improving subsequent cell isolation and antibody staining.
1. Description Myelin removal is therefore recommended prior to cell sorting or
1.1 Principle of the MACS® Separation antibody staining.
1.2 Background information
1.3 Applications
1.3 Applications
● Removal of myelin debris from single-cell suspensions from
1.4 Reagent and instrument requirements human, mouse, or rat during sample preparation significantly
2. Protocol improves efficiency of cell separations and antibody stainings.
2.1 Sample preparation ● Use prior to isolation or staining of neural cell types from mouse
2.2 Magnetic labeling (older than P7) brain tissue by using the respective MicroBeads
or fluorochromes.
2.3 Magnetic separation
3. Examples of separations using the Myelin Removal Beads 1.4 Reagent and instrument requirements
4. References ● Buffer: Prepare a solution containing phosphate-buffered
saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and
2 mM EDTA by diluting MACS BSA Stock Solution (# 130-
1. Description 091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-
222). Keep buffer cold (2−8 °C). Degas buffer before use, as air
Components 2 mL Myelin Removal Beads, human, mouse, bubbles could block the column.
rat ▲  Note: EDTA can be emitted in case of sensitive target cells. BSA can be replaced
Capacity For 2×10⁹ total cells, up to 200 separations. by other proteins such as appropriate serum albumin, appropriate serum, or fetal
bovine serum (FBS). Buffers or media containing Ca 2+ or Mg 2+ are not recommended
Product format Myelin Removal Beads are supplied in buffer for use.
containing stabilizer and 0.05% sodium azide.
● MACS Columns and MACS Separators: Myelin can be depleted
Storage Store protected from light at 2−8 °C. Do not with the use of LD Columns. Depletion can also be performed
freeze. The expiration date is indicated on the by using the autoMACS Pro or the autoMACS Separator.
vial label.
▲  Note: Column adapters are required to insert certain columns into the
VarioMACS™ or SuperMACS™ Separators. For details see the respective MACS
1.1 Principle of the MACS® Separation Separator data sheet.

First, the myelin debris is magnetically labeled with MicroBeads, ● (Optional) Neural Tissue Dissociation Kit (T) (# 130-093-231),
the Myelin Removal Beads. Then, the cell suspension is loaded onto Neural Tissue Dissociation Kit (P) (# 130-092-628), or Neural
a MACS® Column, which is placed in the magnetic field of a MACS Tissue Dissociation Kit – Postnatal Neurons (# 130-094-802)
Separator. The magnetically labeled myelin is retained within the for the preparation of single-cell suspensions from neural
column. The unlabeled cells run through; this cell fraction is thus tissues.
depleted of myelin. ● (Optional) gentleMACS™ Dissociator (# 130-092-235)
1.2 Background information ● (Optional) Pre-Separation Filters (# 130-041-407) to remove
cell clumps.
Myelin, a specialized membrane, ensheathes and insulates axons
in the peripheral and central nervous system. In mice and rats,
myelination begins around birth in the spinal cord and is completed 2. Protocol
in the brain during the first postnatal month.¹ In humans, myelin
formation starts during the second half of fetal life in the spinal 2.1 Sample preparation
cord, peaks during the first year postnatally and can continue until For the preparation of single-cell suspensions from neural tissues
twenty years of age.² refer to the data sheet of the Neural Tissue Dissociation Kit (T)
When myelin containing neural tissue is dissociated, large (# 130-093-231), the Neural Tissue Dissociation Kit (P) (# 130-092-
quantities of myelin debris are generated. Magnetic cell isolation 628), which can both be used in combination with the gentleMACS
and immunostaining of dissociated cells are considerably impaired Dissociator (# 130-092-235), or the Neural Tissue Dissociation
by myelin particles.³,⁴ So far, sucrose solution⁵,⁶ has been used for Kit – Postnatal Neurons (# 130-094-802).
the elimination of myelin but leads to unspecific cell loss.
140-002-714.03

Myelin Removal Beads allow for the specific removal of myelin


debris from single-cell suspensions during sample preparation,

Miltenyi Biotec GmbH Miltenyi Biotec Inc.


Friedrich-Ebert-Straße 68, 51429 Bergisch Gladbach, Germany 2303 Lindbergh Street, Auburn, CA 95602, USA
Phone +49 2204 8306-0, Fax +49 2204 85197 Phone 800 FOR MACS, +1 530 888 8871, Fax +1 530 888 8925
macs@miltenyibiotec.de macs@miltenyibiotec.com
www.miltenyibiotec.com
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Order no. 130-094-544

6. Wash cells by adding 10× the labeling volume of buffer and


centrifuge at 300×g for 10 minutes. Aspirate supernatant
2.2 Magnetic labeling
completely.
7. Resuspend in 500 µL of buffer per each LD Column.
▲ Work fast, keep cells cold, and use pre-cooled solutions. This will
prevent capping of antibodies on the cell surface and non-specific ▲  Note: For example, if you need 6 LD Columns according to the table, resuspend
in 3000 µL of buffer (6×500 µL) and apply 500 µL to each LD Column.
cell labeling.
8. Proceed to magnetic separation (2.3).
▲ The older the mouse or rat the more myelin is present. Therefore,
the volumes of buffer and MicroBeads depend on the age and should
be adjusted according to the following table.
2.3 Magnetic separation
P17– P25–
Mouse or rat <P10 P11–P16 >P30
P24 P30
▲ Choose the appropriate number of LD Columns according to the
Any age
Human – – – – 0.5 g of number of total cells. For details see table in section 2.2.
tissue
▲ Always wait until the column reservoir is empty before proceeding
Volume of buffer per to the next step.
135 µL 540 µL 1080 µL 1620 µL 2250 µL
whole mouse brain
Volume of Myelin ▲ If minimal cell loss is desired, the use of LS Columns or the
Removal Beads per 15 µL 60 µL 120 µL 180 µL 250 µL autoMACS Pro Separator program “Depletes” is recommended.
whole mouse brain This will result in less efficient depletion of myelin debris.
1
Number of LD
Columns required
(for up
1 2 3 4
Depletion with LD Columns
to 5
(500 µL/Column)
brains) 1. Place LD Column in the magnetic field of a suitable MACS
P: postnatal day Separator. For details see LD Column data sheet.

▲  Note: For example, when using three P18 mouse brains, use 3240 µL of buffer 2. Prepare column by rinsing with 2 mL of buffer.
(3×1080 µL) and 360 µL Myelin Removal Beads (3×120 µL). After the washing
step resuspend in 3000 µL of buffer (6×500 µL) because you need 6 LD Columns
3. Apply cell suspension onto the column.
(2 per whole brain) and apply 500 µL of your suspension to each LD Column. 4. Collect unlabeled cells that pass through and wash
When working with dissected tissue pieces instead of whole mouse brain, column with 2×1 mL of buffer. Collect total effluent;
estimate the proportion of the tissue piece in relation to a whole brain and
this is the unlabeled cell fraction. Perform washing
divide the volumes for buffer and Myelin Removal Beads by that factor.
For example, a cerebellum corresponds approximately to a quarter of a whole steps by adding buffer two times. Only add new buffer when
brain, therefore divide volumes of buffer and Myelin Removal Beads by a the column reservoir is empty.
factor of 4. When using only the cerebella from the three P18 mouse brains in
the example above, use 810 µL of buffer (3240 µL:4) and 90 µL Myelin Removal
Beads (360 µL:4). After washing, resuspend in 1000 µL and apply 500 µL of your Magnetic separation with the autoMACS® Pro Separator or the
suspension to each LD Column. autoMACS® Separator
For human (any age):
For a single-cell suspension from a human sample of any age use 450 µL of ▲ Refer to the respective user manual for instructions on how to use
buffer and 50 µL Myelin Removal Beads per 0.5 g of tissue. the autoMACS® Pro Separator or the autoMACS Separator.
▲ For optimal performance it is important to obtain a single‑cell ▲ Buffers used for operating the autoMACS Pro Separator or the
suspension before magnetic labeling. Pass cells through 30 µm autoMACS Separator should have a temperature of ≥10 °C.
nylon mesh (Pre-Separation Filters, # 130-041-407) or appropriate ▲ Program choice depends on the isolation strategy, the strength
mesh size depending on target cells to remove cell clumps which of magnetic labeling, and the frequency of magnetically labeled
may clog the column. Moisten filter with buffer before use. cells. For details refer to the section describing the cell separation
programs in the respective user manual.
▲ The recommended incubation temperature is 2–8 °C. Working
on ice may require increased incubation times. Higher temperatures Magnetic separation with the autoMACS® Pro Separator
and/or longer incubation times may lead to non-specific cell 1. Prepare and prime the instrument.
labeling.
2. Apply tube containing the sample and provide tubes for
1. Determine cell number. collecting the labeled and unlabeled cell fractions. Place
sample tube in row A of the tube rack and the fraction
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate collection tubes in rows B and C.
supernatant completely. 3. For a standard separation choose the following program:
3. Resuspend cell pellet in volume of buffer as according to table Depletion: “Depl05”
above. Collect negative fraction in row B of the tube rack.
4. Add volume of Myelin Removal Beads as according to table
above.
140-002-714.03

5. Mix well and incubate for 15 minutes in the refrigerator


(2−8 °C).

Unless otherwise specifically indicated, Miltenyi Biotec


products and services are for research use only and not for
diagnostic or therapeutic use.

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Magnetic separation with the autoMACS® Separator 4. References Order no. 130-094-544

1. Prepare and prime the instrument. 1. Simons, M. and Trotter, J. (2007) Wrapping it up: the cell biology of myelination.
Curr. Opin. Neurobiol. 17: 533–40.
2. Apply tube containing the sample and provide tubes for 2. Baumann, N. and Pham-Dinh, D. (2001) Biology of Oligodendrocyte and
collecting the labeled and unlabeled cell fractions. Place Myelin in the Mammalian Central Nervous System. Physiol. Rev. 81: 871–927.
sample tube at the uptake port and the fraction collection 3. Pfenninger, C.V. et al. (2007) CD133 Is Not Present on Neurogenic Astrocytes
tubes at port neg1 and port pos1. in the Adult Subventricular Zone, but on Embryonic Neural Stem Cells,
Ependymal Cells, and Glioblastoma Cells. Cancer Res. 67: 5727–5736.
3. For a standard separation choose the following program:
4. Tham, C.S. et al. (2003) Microglial activation state and lysophospholipid acid
Depletion: “Depl05” receptor expression. Int. J. Dev. Neurosci. 21: 431–43.
Collect negative fraction from outlet port neg1. 5. Johansson, C.B. et al. (1999) Identification of a neural stem cell in the adult
mammalian central nervous system. Cell 96: 25–34.
6. Szuchet, S. et al. (1980) Separation of ovine oligodendrocytes into two distinct
3. Example of myelin depletion using Myelin bands on a linear sucrose gradient. J. Neurosci. Methods 3: 7–19.

Removal Beads 7. Reiß, S. et al. (2008) Removal of myelin debris after tissue dissociation optimizes
immunomagnetic sorting of cells from postnatal brain. SfN Neuroscience
Myelin debris is abundant in single-cell suspensions Meeting, November 15–19, 2008, Washington DC, USA, 38th annual meeting
of the Society for Neuroscience, poster no. 623.9.
Mouse brains were dissociated using the Neural Tissue Dissociation
Kit (P) (# 130-092-628). Flow cytometrical analysis of the resulting
single-cell suspensions shows the distribution of cells and myelin All protocols and data sheets are available at www.miltenyibiotec.com.
debris (A). Single-cell suspensions from postnatal day P22 mouse
Warnings
brains consist of large amounts of myelin membrane fragments, Reagents contain sodium azide. Under acidic conditions sodium azide yields
other cellular debris and only 4% cells. Debris outside the selected hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with
gates are positive for anti-O1, anti-O4, and anti-GalC that recognize running water before discarding. These precautions are recommended to avoid
glycolipids specifically contained in myelin membranes (B). Myelin deposits in plumbing where explosive conditions may develop.
Removal Beads used with LD Columns and MidiMACS Separator
efficiently remove myelin debris (C). Warranty
The products sold hereunder are warranted only to be free from defects in workmanship
and material at the time of delivery to the customer. Miltenyi Biotec GmbH
Before depletion
makes no warranty or representation, either expressed or implied, with respect to
the fitness of a product for a particular purpose. There are no warranties, expressed
A) Cells and myelin debris B) O1-, O4-, GalC-positive events or implied, which extend beyond the technical specifications of the products.
Miltenyi Biotec GmbH’s liability is limited to either replacement of the products or
refund of the purchase price. Miltenyi Biotec GmbH is not liable for any property
damage, personal injury or economic loss caused by the product.
Side scatter

Side scatter

autoMACS and MACS are registered trademarks and gentleMACS, MidiMACS,


SuperMACS, and VarioMACS are trademarks of Miltenyi Biotec GmbH.

Copyright © 2009 Miltenyi Biotec GmbH. All rights reserved.

Forward scatter Forward scatter


After depletion

C) After myelin removal


Side scatter

Forward scatter
140-002-714.03

Unless otherwise specifically indicated, Miltenyi Biotec


products and services are for research use only and not for
diagnostic or therapeutic use.

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