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 O4-positive oligodendrocytes using the Anti-O4 MicroBeads, human, mouse, rat. Follow the protocol of the kit data sheet.

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Anti-O4 MicroBeads, human, mouse, rat

Notes:

• The recommended antibody dilution for labeling cells is 1:11 for up to 1×10⁷ cells/100 μL of PB buffer.
• Volumes given below are for up to 1×10⁷ nucleated cells. When working with fewer than 1×10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g., for 2×10⁷ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

1. Analyze 100 µL of the positive and 100 µL of the negative fractions. Optionally, also include 20 µL of the original fraction in the analysis.
2. Resuspend up to 1×10⁷ nucleated cells per 100 μL of PB buffer (see "Things to prepare in advance of cell isolation and cell culture").
3. Add 10 μL of Anti-O4-APC.
4. Mix well and incubate for 10 minutes in the dark in the refrigerator (2–8 °C).
   ▲ Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
5. Wash cells by adding 1 mL of PB buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
6. Resuspend cell pellet in a suitable amount of PB buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10. 
   

1. Plate 5×10⁴ cells in 50 µL of prepared medium as a drop in the middle of each well of a coated 24-well plate (see "Things to prepare in advance for cell isolation and cell culture”).
2. Let cells settle 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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