Application protocol

Isolation and cultivation of oligodendrocyte precursor cells from neonatal mouse brain

This application protocol describes the separation of oligodendrocyte precursor cells based on the expression of CD140a, or platelet-derived growth factor receptor alpha (PDGFRα), which is broadly expressed in embryonic tissue, various malignancies and embryonic stem cell-derived cardiomyogenic cells1,2, and specifically expressed by oligodendrocyte precursor cells that differentiate into myelinating oligodendrocytes.3–5 The isolation protocol has been tested on dissociated postnatal CD1 mouse brain tissue derived from animals younger than postnatal day eight (P8). In principle, cell isolation is also possible from older mice, but purity of the positive fraction might be lower due to a lower frequency of CD140a (PDGFRα)-positive cells after tissue dissociation. For optimal results, we recommend using the Neural Tissue Dissociation Kit (P).

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol.  These products are for research use only.

For brain tissue dissociation

  • Neural Tissue Dissociation Kit (P) (#130-092-628) or Neural Tissue Dissociation Kit (T) (# 130-093-231)
  • Hanks' Balanced Salt Solution (HBSS) without Ca2+ and Mg2+
  • HBSS with Ca2+ and Mg2+
  • gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or
    gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237, # 130-096-334)
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.
  • (Optional) Beta-mercaptoethanol, 50 mM
  • MACS® SmartStrainers (70 µm) (# 130-090-753) in combination with an incubator at 37 °C
  • MACSmix™ Tube Rotator (#130-098-753)
  • 50 mL tubes

For cell isolation and flow cytometry analysis

  • CD140a (PDGFRα) MicroBead Kit, mouse (#130-101-502; small size 130-101-547)
  • Pre-Separation Filters (70 µm) (#130-095-823)
  • Fluorochrome-conjugated Labeling Check Reagents to stain labeled cells for flow cytometry analysis, e.g., Labeling Check Reagent-APC (130-098-892), or Labeling Check Reagent-PE (# 130-095-228). Learn more about our antibodies and dyes.
    Note: The use of CD140a antibodies, clone APA5, is not recommended for analysis of cells that are labeled with CD140a (PDGFRα) MicroBeads.
  • Propidium Iodide Solution (#130-093-233) or 7-AAD for flow cytometric exclusion of dead cells.
  • (Optional) MACSQuant® Analyzer 10 (# 130-096-343)
  • PB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2 and 0.5 % bovine serum albumin (BSA) by diluting MACS BSA Stock Solution (# 130-091-376) 1:20 with PBS. Keep buffer cold (2-8 °C). Degas buffer before use, as air bubbles could block the column.
    Note: BSA can be replaced by other proteins such as mouse or rat serum albumin, mouse serum, or fetal bovine serum (FBS).
  • MACS Columns and MACS Separators: CD140a (PDGFRα)+ cells can be enriched using LS or MS Columns. Positive selection can also be performed using the autoMACS Pro Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×1072×107MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
LS2×1074×107

MidiMACS™, QuadroMACS™,

VarioMACS, SuperMACS II

autoMACS5×1071×108autoMACS Pro
Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet. 

For cell culture

  • Double-distilled water (ddH₂O)
  • Imaging Plate CG 1.5 (24 well) (# 130-098-263)
  • MACS Neuro Medium (# 130-093-570)
  • MACS NeuroBrew®-21 (# 130-093-566)
  • 200 mM L-glutamine
  • Poly-L-lysine (0.01%)
  • Penicillin/streptomycin
  • Human PDGF-AA (# 130-093-977)
  • Human FGF-2 (# 130-093-837) 

For immunocytochemical staining of cultured cells

  • Anti-O4 pure, human, mouse, rat (# 130-115-810) and anti-mouse IgM secondary antibody
  • Staining buffer: Prepare a solution containing autoMACS Running Buffer (# 130-091-221) with FcR Blocking Reagent, mouse (# 130-092-575) in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.
  • Phosphate-buffered saline (PBS)
  • autoMACS Running Buffer (# 130-091-221)
  • 2% paraformaldehyde (PFA) for fixation
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Protocol


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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.