Intracellular Staining

• Prepare target cells in cold PBS. 1 x 106 cells/sample is enough for analysis.
• If staining a cell surface receptor, stain with antibody in 100 mL of PBS before fixing cells.
• Wash 2X with PBS. Stain with secondary antibody if necessary.
• Wash 2X with PBS.
• Fix cells in 0.5 mL of 4% paraformaldehyde solution and incubate at 22 ° C for 20 minutes in the DARK.
• Vortex gently at intermittent times to avoid clumping of cells.
• Wash the fixed/stained cells 2X with cold PBS.
• Add 2 mL of blocking buffer and block for 20 minutes at 22 ° C.
• Add appropriate amount of conjugated anti-cytokine Mab.
• Incubate at 22 ° C for 20 minutes in the DARK.
• Wash once with PBS containing 0.1% saponin, remove supernatant and resuspend the cell pellet with 0.5 mL 2% formaldehyde solution.
• Samples should be at a minimum concentration of 5 X 10 6 cells/mL
• Send to MDS for FACS analysis