Analyzing Efficacy of Small Molecule Blocker of TNF-a in Sorted CD4 T cells by using PE Conjugated anti-TNF-a Antibody and Flow Cytometry

Protocol Overview

Human CD4+ T cells were sorted from whole blood using negative selection bead method applying a magnetic bead column protocol.Sorted cells were stimulated with PMA (20ng/ml) and Ionomycin( 1ug/ml)for 4 hours, with Brefeldin A (GolgiPlug) added to the media. Cells were washed twice with PBS and the following protocol was followed for intracellular staining for TNF-a and IFN-g:a) Thoroughly resuspend cells and add 100uL per well for microwell plates (or 250 uL for tubes) of Fixation/Permeabilization solution for 20 minutes at 4Cb)Wash cells two times in 1X BD Perm/Wash™ buffer (e.g., 1 mL/wash for staining in tubes and 250 uL/wash final volume for staining in microwell plates) .c)Thoroughly resuspend permeabilized cells in 50 uL BD Perm/Wash™ buffer containing a pre-determined optimal concentration of a fluorochrome-conjugated anti-cytokine antibody(PE anti-TNF-a and APC anti-IFN-g) or appropriate negative control. Incubate at 4C for 30 minutes in the dark.d)Wash cells twice with 1X BD Perm/Wash™ buffer (1 mL/wash for staining in tubes and 250 uL/wash final volume for staining in microwell plates) and resuspend in Staining Buffer prior to flow cytometric analysis.