Application protocol

Isolation and cultivation of O4-positive oligodendrocytes from neonatal mouse brain

This application protocol describes the isolation of highly purified and viable oligodendrocytes from neonatal mouse brain tissue. Brain tissue from mice younger than P8 is dissociated into a single-cell suspension and Anti-O4 MicroBeads are used to isolate oligodendrocytes. The O4 antigen, a sulfatide of the glycosphingolipid class is a marker for oligodendrocytes. During oligodendrocyte development, O4 expression begins on late oligodendrocyte progenitors that are A2B5-positive. While A2B5 expression disappears, O4 continues to be expressed. O4 expression is also found in Schwann cells. The isolation of O4+ cells leads to highest purities if P3–P7 (postnatal day 3–7) rodents are used. Mouse brain tissue derived from P3–P7 CD-1® mice contains approximately 5–10% O4+ cells.


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

  • Anti-O4 MicroBeads, human, mouse, rat (#130-094-543; small size 130-096-670)
  • FcR Blocking Reagent, mouse (# 130-092-575) or FcR Blocking Reagent, human (#130-059-901)
  • Pre-Separation Filters (70 µm) (#130-095-823)
  • Fluorochrome-conjugated Anti-O4 antibodies for flow cytometry analysis, e.g., Anti-O4-PE (# 130-095-887) or Anti-O4-APC (# 130-095-891). Learn more about our antibodies and dyes.
  • 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: O4+ cells can be enriched using LS or MS Columns. Positive selection can also be performed using the autoMACS® Pro Separator or the MultiMACS™ Cell24 Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×1072×107MiniMACS™, OctoMACS™
LS2×1074×107MidiMACS™, QuadroMACS™
autoMACS5×1071×108autoMACS Pro
Multi-242×1074×107MultiMACS Cell24

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