Tnfα bv711 clone mab11

Levels of Th cells were determined as the percentage of CD4⁺ T cells that produce these cytokines following in vitro stimulation with PMA & Ionomycin. PBMCs, isolated as described above, were resuspended to 1×10⁶ cells/mL in RPMI 1640 medium plus 10% FBS (R10). Cells were then incubated for 5h at 37°C in a medium with 1 × Cell Stimulation Cocktail (containing 81 nM PMA and 1.34 nM ionomycin plus protein transport inhibitors, Ebioscience, San Diego, CA, USA). Following incubation, the cells were washed and surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV510 for 30 minutes in the dark at room temperature, followed by fixation and permeabilization. After permeabilization, cells were stained with IFN-γ-AF700 (clone 4S.B3), IL-22-FITC (clone 22URTI), IL-9-PE (clone MH9D1), IL-27-APC (clone ebic6), IL-10-PE-CY7 (clone JES3-9D7, Ebioscience), TNF-α-BV711 (clone MAb11), IL-17-BV421 (clone BL168), IL-2-BV650 (clone MQ1-17H12, BioLegend), or GM-CSF-PE-CF594 (clone BVD2-21C11, BD Biosciences) antibodies for 30 minutes in the dark at room temperature. Following staining, cells were washed and acquired on an LSRFortessa.