Immunofluorescent Staining of Whole Blood
Add predetermined optimum concentrations of desired fluorochrome conjugated, biotinylated, orpurified primary antibodies to 100 μl of anti-coagulated whole blood.
Incubate at room temperature for 15-20 minutes in the dark.
Dilute 10X Red Blood Cell (RBC) Lysis Buffer (BioLegend Cat.
No.
420301) to 1X working concentrationwith DI water.
Warm to room temperature prior to use.
Add 2 ml of 1X RBC lysis solution to wholeblood/antibody mixture.
Incubate at room temperature for 10 minutes.
Centrifuge at 350 X g for 5 minutes, discard the supernatant.
Wash 1X with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes.
If using a purified primary antibody, resuspend pellet in residual buffer and add a previously determinedoptimum concentration of anti-species immunoglobulin fluorochrome conjugated secondary antibody(e.g.
FITC anti-mouse Ig) and incubate in the dark for 15-20 minutes.
Wash 1X with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes.
Resuspend cells in 0.5 ml Cell Staining Buffer or 0.5 ml 2% paraformaldehyde-PBS fixation buffer.
Analyze with a Flow Cytometer.
