Intracellular Cytokine Staining Protocol

Intracellular Staining:
7. Resuspend fixed/permeabilized cells in residual Permeabilization Wash
Buffer and add a predetermined optimum concentration of fluorochrome
conjugated antibody of interest (e.g. anti-IFN-γ-PE) or an appropriate negative
control for 20 minutes in the dark at room temperature.
8. Wash 2x with 2 ml of Permeabilization Wash Buffer and centrifuge at 350 x g
for 5 minutes.
9. If primary intracellular antibody is biotinylated, it will be necessary to perform
fluorochrome conjugated Streptavidin incubations and subsequent
washes in Permeabilization Wash Buffer.
10. Resuspend fixed and intracellularly labeled cells in 0.5 ml Cell Staining Buffer
and analyze with appropriate controls.
Note: To confirm specific anti-cytokine staining, a blocking experiment is recommended
in which cells are fixed/permeabilized then preincubated with an
excess amount of unlabeled anti-cytokine antibody and/or the recombinant
cytokine of interest is preincubated with fluorochrome-conjugated anticytokine
antibody before its addition to the cells.