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.

Use the Neural Tissue Dissociation Kit (P) or Neural Tissue Dissociation Kit (T) to dissociate brain tissue and prepare a single-cell suspension. Follow the protocol of the kit data sheet.

Download data sheet

Neural Tissue Dissociation Kits

Isolate the ACSA-2-positive astrocytes from the single-cell suspension using the Anti-ACSA-2 MicroBead Kit, mouse. Follow the protocol of the kit data sheet.

Download data sheet

Anti-ACSA-2 MicroBead Kit, mouse

Notes:

• The recommended antibody dilution for labeling cells is 1:10 for up to 1×10⁶ cells/50 μL of PB 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. Use 100 μL of the ACSA-2⁺ fraction for analysis. Optionally, also analyze 100 μl of the negative fraction and 20 μL of the original fraction.
2. Resuspend up to 1×10⁶ nucleated cells per 45 μL of PB buffer (see "Things to prepare in advance of cell isolation and cell culture").
   ▲ Note: Always use freshly prepared buffer.
   
3. Add 5 μL of Anti-ACSA-2-APC, mouse antibodies.
4. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C) in the dark.
   ▲ 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 pre-warmed prepared medium as a drop in the middle of each well of a coated 24-well plate (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 medium every other day.
   

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 primary antibody in staining buffer to the cells with a final concentration of 1–5 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 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.
    

1. G. Kantzer, C. et al. (2017) Glia 65: 990–1004.
2. Batiuk, M.Y. et al. (2017) J Biol. Chem. 292: 8874-8891
   

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