For the stimulation of cells, various in vitro methods have been reported for stimulating cells to produce cytokines. Polyclonal activators have been particularly useful for inducing cytokine-producing cells. These activators include the following: concanavalin A, lipopolysaccharide, phorbol esters plus calcium ionophore or ionomycin, phytohaemaglutinin, staphylococcus enterotoxin B, and monoclonal antibodies directed against subunits of the TCR/CD3 complex (with or without antibodies directed against costimulatory receptors, such as CD28).

1. Fix and Permeabilize Cells

        a. Thoroughly resuspend cells in 100 µL of  BD Cytofix/Cytoperm  solution per well for microwell plates (or 250 µL for tubes) and incubate for 20 min. at 4°C.  Note: Cell aggregation can be avoided by vortexing prior to the addition of the BD Cytofix/Cytoperm™ solution.

        b. Wash cells two times in a buffer that contains a cell permeabilizing agent such as saponin (BD Perm/Wash™ buffer, Cat. 554723, which can be used as the wash buffer and as the antibody diluent).

2. Stain for Intracellular Cytokines

        a. Thoroughly resuspend fixed/permeabilized cells in 50 µL of a saponin-containing buffer (e.g., BD Perm/Wash™ buffer) containing a pre-determined optimal concentration of a fluorochrome-conjugated anti-cytokine antibody or appropriate negative control. Incubate at 4°C for  30 minutes in the dark.  Note: Because saponin-mediated cell permeabilization is a reversible process, it is important to keep the cells in the presence of saponin during intracellular cytokine staining.

        b. Wash cells 2 times with saponin-containing buffer (e.g, BD Perm/Wash™ buffer) and resuspend in staining buffer prior to flow cytometric analysis.

        Note: Both the BD Cytofix/Cytoperm™ (Cat. No. 554722) (固定液)and BD Perm/Wash buffer (Cat. No. 554723 （破膜液）) are included in the  Fixation/Permeabilization Solution Kit (Cat. No. 554714)（固定和破膜液） as well as the Fixation/Permeabilization Solution Kit with BD GolgiStop (containing monensin莫能菌素); Cat. No. 554715) and Fixation/Permeabilization Solution Kit with GolgiPlug (containing brefeldin A , BFA); Cat.  No. 555028).