Microglia FACS staining after isolation (from UCSD)

Resuspend cells staining buffer (300µL of HBSS+EDTA+BSA).

• Resuspend in a 15-ml falcon tube.

Add Fc Block (~1:100 dilution) and incubate for 0h 15m 0s at 4°C or in the fridge.

• Add this to cells resuspended in the HBSS.

Take ~5% of cells for unstained control, put On ice.

Add antibodies (CD11b, CD45, 1:100 dilution) for 20-30 min at 4°C.

Add directly to cells in the FC block.

Mix by tapping tube; do not vortex.

Add DAPI, 0h 0m 45s, then dilute with HBSS+BSA+EDTA.

Add 1:1000 dilution.

Add to unstained cells.

Put 70-μm filter on top of a FACS test tube and filter resuspended pellet into the tube.

Push filter hard onto tube.

Centrifuge for 400x g.

Prepare collection tubes during spin time.

• Coat FACS tubes with 1mL of staining buffer to prevent cells from sticking to walls of tubes.
• Invert tube several times.

Vacuum out supernatant.

• Remove most of supernatant; not all.

Resuspend pellet in 500µL to 1000µL staining buffer.