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). 
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). 
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.
Wash with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes. (2/2). 
Wash with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes. (2/2).
