Isolation and cultivation of microglia from neonatal mouse or rat brain 

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: DMEM containing 10% FBS, 1% penicillin/streptomycin, and 2 mM L-glutamine.

(Optional) Add 20 ng/mL M-CSF (#130-094-129) to enhance microglia growth and survival. 
 

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

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Neural Tissue Dissociation Kits

Isolate microglia from the single-cell suspension using either the CD11b (Microglia) MicroBeads, human and mouse or the CD11b/c (Microglia) MicroBeads, rat. Follow the protocol of the corresponding kit data sheet.

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CD11b (Microglia) MicroBeads, human and mouse

CD11b/c (Microglia) MicroBeads, rat

Notes:

• The recommended antibody dilution for labeling cells is 1:10 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. Use 100 μL of the CD11b⁺ fraction for analysis. Optionally, also analyze 100 μl of the negative fraction and 20 μL of the original fraction.
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").
   ▲ Note: Always use freshly prepared buffer.
3. Add 2 µL of CD11b- APC and CD45-FITC.
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.
    

Notes:

• The recommended antibody dilution for labeling cells is 1:10 for up to 1×10⁶ cells/50 μL of 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 CD11b/c⁺ 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 buffer.
   ▲ Note: Always use freshly prepared buffer.
3. Add 5 µl CD11b/c-FITC and CD45-APC.
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 buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
6. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry, e.g., using the MACSQuant Analyzer 10.
    

1. Plate 1×10⁵ 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 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: 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.
   ▲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 5–10 μg/mL and incubate at room temperature in the dark for 10 minutes.
   When working with CD68 antibodies: Incubate at 2–8 °C overnight in the dark
7. Wash cells 3× with autoMACS Running Buffer.
8. Add a corresponding secondary antibody in staining buffer to the cells.
   When working with CD11b antibodies: Incubate at room temperature in the dark for 10 minutes.
   When working with CD68 antibodies: Incubate at room temperature in the dark for 1 hour.
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.