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 μl 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.

To analyze the results, call Cytometry Research for a FACS analysis appointment.