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 1:20 with PBS. Keep buffer cold (2−8 °C). Prepare fresh on the day of use and degas the buffer, as air bubbles could block the column.

▲ Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS).

Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.

Prepare the following cell culture medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine, 10 ng/mL Human PDGF-AA, and 10 ng/mL Human FGF-2.

Dissociate mouse neonatal brain using the Neural Tissue Dissociation Kit (P) or Neural Tissue Dissociation Kit (T). Follow the protocol of the kit data sheet.

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Neural Tissue Dissociation Kits

Isolate oligodendrocyte precursor cells using the CD140a (PDGFRα) MicroBead Kit, mouse. Follow the protocol of the kit data sheet.

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CD140a (PDGFRα) MicroBead Kit, mouse

To stain and analyze the isolated oligodendrocyte precursor cells by flow cytometry, follow the protocol of the Labeling Check Reagent.
 

1. Plate 5×10⁴ cells in 50 µL of prepared medium as a drop in the middle of each well of a 24-well plate that has been coated overnight (see "Things to prepare in advance of cell isolation and cell culture”).
2. Let the cells settle down for 30 minutes at 37 °C in the incubator.
3. Carefully add 450 µL of prepared medium to each well.
4. Maintain the culture by replacing 50% of prepared medium every other day.
   ▲Note: If oligodendrocytes are cultivated for longer periods, e.g., 2 weeks, we recommend increasing FGF-2 and PDGF-AA concentrations to 20 ng/mL instead of 10 ng/mL.
   

1. Wash cells 3× with PBS.
2. Fix cells with 2% PFA for 10 minutes at room temperature.
3. Wash cells 3× with PBS.
   ▲Note: Fixed cells can be stored in azide-containing buffer at 2–8 °C for up to 1 week.
4. Add staining buffer (see "Things to prepare in advance") and incubate for 10 minutes at room temperature.
5. Discard staining buffer.
6. Add Anti-O4 pure antibody in staining buffer to the cells with a final concentration of 5–10 μg/mL and incubate at room temperature in the dark for 10 minutes.
7. Wash cells 3× with autoMACS Running Buffer.
8. Add a corresponding secondary antibody (anti-mouse IgM) in staining buffer to the cells and incubate at room temperature in the dark for 10 minutes.
9. Wash cells 3× with autoMACS Running Buffer.
   ▲Note: For co-staining with additional antibodies repeat steps 6–9.
10. Store cells in autoMACS Running Buffer.
11. Cells are now ready for immunofluorescence microscopy.
    ▲Note: Samples can be stored at 2–8 °C in the dark for up to one week
    ▲Note: When working with cells cultured on coverslips, the coverslips need to be mounted onto slides before imaging.
    

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