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).
Procedure for Using BD Perm/Wash™ buffer:
1. Fix cells using a buffer containing paraformaldehyde, or use BD Cytofix/Cytoperm™ solution (Cat. No. 554722)
2. Permeabilize fixed cells by washing 2 times in 1X BD Perm/Wash buffer (Cat. No. 554723) (e.g., 1 ml/wash for staining in tubes and 250 µl/wash final volume for staining in microwell plates). Incubate for 15 minutes in 1X BD Perm/Wash buffer (the 15 minute incubation can be omitted if BD Cytofix/Cytoperm is used for fixing cells). Pellet cells.
a. Dilute 10X BD Perm/Wash buffer in distilled H2O to make a 1X solution prior to use.
3. Stain for Intracellular Cytokines
a. Thoroughly resuspend fixed/permeabilized cells in 50 µl of 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.
b. Wash cells 2 times with 1X BD Perm/Wash buffer (1 ml/wash for staining in tubes and 250 µl/wash final volume for staining in microwell plates) and resuspend in staining buffer prior to flow cytometric analysis.
Note: Both the BD Perm/Wash buffer (Cat. No. 554723) and the BD Cytofix/Cytoperm solution (Cat. No. 554722) are included in the BD Cytofix/Cytoperm Kit (Cat. No. 554714) as well as the BD Cytofix/Cytoperm Plus Kit with BD GolgiStop™ protein transport inhibitor (containing monensin; Cat. No. 554715) and BD Cytofix/Cytoperm Plus™ Kit with BD GolgiPlug™ protein transport inhibitor (containing brefeldin A; Cat. No. 555028).
Warning: The BD Perm/Wash solution contains saponin and sodium azide which is known to be toxic. Avoid contact with skin, eyes and mucous membranes.