DPBS/BSA buffer: Prepare a solution containing Dulbecco’s phosphate-buffered saline (DPBS) with Ca²⁺ and Mg²⁺ and 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution 1:20 with DPBS. Keep buffer cold (2–8 °C). Prepare fresh on the day of use and degas the buffer, as air bubbles could block the column.

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

For cell culture, prepare the following medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.

Dissociate mouse neonatal brain using the Neural Tissue Dissociation Kit – Postnatal Neurons. Follow the protocol of the kit data sheet.
 

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Neural Tissue Dissociation Kit – Postnatal Neurons

Isolate neurons from the single-cell suspension using the Neuron Isolation Kit, mouse. Follow the protocol of the kit data sheet.
 

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Neuron Isolation Kit, mouse

Notes:

• The recommended antibody dilution for labeling of cells is 1:10 for up to 1×10⁶ cells/50 μL of DPBS/BSA buffer.
• Volumes given below are for up to 1×10⁶ nucleated cells. When working with fewer than 1×10⁶ 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 2×10⁶ 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 analyze 20 μL of the original fraction.
2. Centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
3. Resuspend up to 1×10⁶ nucleated cells per 45 μL of DPBS/BSA buffer (see "Things to prepare in advance of tissue dissociation and cell culture").
4. Add 5 μL of Anti-Biotin-PE.
5. 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.
6. Wash cells by adding 1 mL of DPBS/BSA buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
7. Resuspend cell pellet in a suitable amount of DPBS/BSA buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.
   

1. Plate 1×10⁵ 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 of tissue dissociation 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. Take off the medium to remove non-attached and dead cells.
   
5. Add 500 μL of prepared medium.
6. Maintain the culture by replacing 50% of medium every other day.
   

Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse (1:10).

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: When working with CD68 antibodies, add 0.2% TRITON X-100 in PBS, incubate for 10 minutes at room temperature, and wash cells 3× with autoMACS® Running Buffer. Do not treat cells with TRITON X-100 before staining with Anti-ACSA-2, Anti-O4, or Anti-PSA-NCAM antibodies.
   ▲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 pure antibody of choice in staining buffer to the cells and incubate in the dark. For incubation temperature and time as well as recommended antibody concentration, refer to the table below.
   
7. Wash cells 3× with autoMACS Running Buffer.
   
8. Add a corresponding secondary antibody in staining buffer to the cells and incubate in the dark. For incubation temperature and time as well as recommended antibody concentration, refer to the table below.
   
9. Wash cells 3× with autoMACS Running Buffer.
   ▲Note: For co-staining with additional antibodies repeat step 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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