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 concentration with 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 determined optimum 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 .
