Harvest and wash cells according to the manufacturer's guidance. 0.5M EDTA is recommended for the adherent cells.
Determine the total cell number and check cell viability. >90% is recommended.
Spin down and resuspend cell samples in an ice-cold suspension buffer. 2%FBS with DPBS is recommended.
Proceed to stain with a viability dye. DNA binding dyes, such as 7-AAD and DAPI are often used as viability dyes for live/dead staining, as they cannot penetrate the cell membrane of live cells.
Stain cells with a viability dye.
Wash cells two times with wash buffer. 2% FBS with DPBS is recommended.
Proceed to blocking when detecting extracellular targets or to fixation and permeabilization for intracellular targets.
Fix the cells in your chosen fixative. 2% paraformaldehyde (PFA), 30 min on ice is recommended.
Wash the cells two times with suspension buffer.
Permeabilize cells by incubating them with a suitable detergent. 0.2% Tween-20 in PBS, 10min in room temperature is recommended.
Wash the cells two times with the suspension buffer.
Block Fc receptors with a blocking buffer. 2% goat serum in DPBS is recommended.
Wash cells two times with the wash buffer.
Proceed to antibody incubation.
Dilute the conjugated primary antibody in the suspension buffer.
Incubate cells in the pre-diluted primary antibody. Incubate in the dark for 1 hour on ice.
Wash the cells two times with the suspension buffer.
Proceed to detection in the flow cytometer as soon as possible.
