Application protocol

Isolation and cultivation of oligodendrocytes from adult mouse or rat brain 

 In this application protocol, we describe a protocol to generate highly purified and viable oligodendrocytes from adult mouse or rat brain tissue. Brain tissue from mice or rats older than P7 is dissociated into single-cell suspensions using the Adult Brain Dissociation Kit. The extracellular matrix is enzymatically digested using the kit components, while the gentleMACS™ Dissociator with Heaters is used for the mechanical dissociation steps during the on-instrument enzyme incubation. After the dissociation, the myelin and cell
debris are removed using the Debris Removal Solution and is followed by an subsequent removal of erythrocytes using the Red Blood Cell Removal Solution. The Anti-O4 MicroBeads are used to isolate oligodendrocytes from the single-cell suspension.

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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.

General reagent and instrument requirements

  • Dulbecco’s phopshate-buffered saline (D-PBS) with calcium, magnesium, glucose, and pyruvate. Keep buffer cold (2-8 °C).
  • D-PBS/BSA buffer: Prepare a solution containing D-PBS anD 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with D-PBS. Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
    Always use freshly prepared buffer. Do not use autoMACS® Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.
    Note: BSA can be replaced by other proteins such as mouse or rat seruM albumin, mouse or rat serum, or fetal bovine serum (FBS).

For brain tissue dissociation

  • Adult Brain Dissociation Kit, mouse and rat (# 130-107-677)
  • gentleMACS™ Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237, # 130-096-334)
  • 35 mm diameter sterile petri dish
  • Sterile scalpel
  • Sterile forceps
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.
  • MACS SmartStrainers (70 µm) (# 130-098-462)
  • 15 mL and 50 mL tubes
  • Centrifuge with swinging bucket rotor

For cell isolation and flow cytometry analysis

  • Anti-O4 MicroBeads, human, mouse, rat (#130-094-543)
  • (Optional) Pre-Separation Filters (70 µm) (#130-095-823)
  • MACS Columns and MACS Separators: O4+ cells can be enriched by using MS Columns. Positive selection can also be performed by using the autoMACS Pro Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSelector
MS1 x10⁷2 x10⁷MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
autoMACS5 x10⁷1 x108autoMACS 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.
  • Anti-O4 antibodies, human, mouse, rat (clone O4 or clone REA576) conjugated to PE or APC. Learn more about our antibodies and dyes.
  • (Optional) Propidium Iodide Solution (#130-093-233) or 7-AAD for flow cytometric exclusion of dead cells.
  • (Optional) MACSQuant Analyzer 10 (# 130-096-343)

For cell culture

  • Double-distilled water (ddH₂O)
  • Imaging Plate CG 1.5 (24 well) (# 130-098-263)
  • MACS Neuro Medium (# 130-093-570) and 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)
  • FcR Blocking Reagent, mouse (# 130-092-575)
  • 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.