Microglia FACS staining after isolation (from UCSD)
rabdelha
Abstract
This protocol details about microglia FACS staining after isolation (from UCSD).
Attachments
437-925.docx
Steps
Protocol
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
1mLof 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.
