Cell Surface Immunofluorescence Staining Protocol
Obtain desired tissue ( e . g .
spleen , lymph node , thymus , bone marrow ) and prepare a single cell suspension in Cell Staining Buffer ( BioLegend Cat .
No .
420201 ) .
Add Cell Staining Buffer up to ~ 15 ml and centrifuge at 350 x g for 5 minutes , discard supernatant .
If necessary ( e . g .
spleen ) , dilute 10X Red Blood Cell ( RBC ) Lysis Buffer ( BioLegend Cat .
No .
420301 ) to 1X working concentration with DI water and resuspend pellet in 3 ml 1X RBC Lysis Buffer .
Incubate on ice for 5 minutes .
Stop cell lysis by adding 10 ml Cell Staining Buffer to the tube .
Centrifuge for 5 minutes at 350 x g and discard supernatant .
Add Cell Staining Buffer up to ~ 15 ml and centrifuge at 350 x g for 5 minutes , discard supernatant .
Count viable cells and resuspend in Cell Staining Buffer at 5 - 10 x 106 cells / ml and distribute 100 μl / tube of cell suspension ( 5 - 10 x 105 cells / tube ) into 12 x 75 mm plastic tubes .
Add appropriately conjugated fluorescent , biotinylated , or purified primary antibodies at predetermined optimum concentrations ( e . g .
anti - CD3 - FITC , anti - CD4 - Biotin , and anti - CD8 - APC ) and incubate on ice for 15 - 20 minutes in the dark .
Wash with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes .
( 1 / 2 ) .
Wash with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes .
( 2 / 2 ) .
If using a purified primary antibody , resuspend pellet in residual buffer and add previously determined optimum concentrations of anti - species immunoglobulin fluorochrome conjugated secondary antibody ( e . g .
FITC anti - mouse Ig ) and incubate on ice in the dark for 15 - 20 minutes .
Wash with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes .
( 1 / 2 ) .
Wash with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes .
( 2 / 2 ) .
Resuspend cell pellet in 0.5 ml of Cell Staining Buffer and add 5 μl ( 0.25 μg ) / million cells of 7 - AAD Viability Staining Solution ( BioLegend Cat .
No .
420403 ) to exclude dead cells .
Incubate on ice for 3 - 5 minutes in the dark .
Analyze with a Flow Cytometer .
