Direct staining
Reagents
Method
Controls required
- Staining buffer (1X PBS + 1 mM EDTA + 2%FBS/BSA)
- Fc blocker
- Antibodies
- PBS + 1 mM EDTA
- Fixable viability dye
Method
- Collect cells and transfer approx. 1 million cell/tube (or well if staining in 96-well plate)
- Wash cell twice in cold PBS (be careful not to discard the cell pellet!)
- Add viability dye diluted in PBS, incubate 15 min on ice in the dark
- Wash cell twice with staining buffer
- Add Fc blocker, incubate 15 min on ice in the dark
- Add extracellular antibodies diluted in staining buffer, incubate 20 min on ice in the dark
- Wash cells twice in cold staining buffer
- Resuspend in staining buffer and go to the facility (keep your cells on ice and in the dark until acquisition)
Controls required
- Unstained sample
- Single-stained controls for compensation (use compensation beads or cells) for each fluorochrome you are using
- Fluorescence minus one controls
- Biological positive and negative controls
