Pacific blue clone ucht1

Fluorochrome conjugated mAbs specific for human CD3 (Pacific Blue, clone UCHT1), human CD20 (FITC, clone L27 and APC H7 clone 2H7), human CD4 (PerCP, clone SK3), human IL17A (APC, clone BL168), TNFα (PE, clone IT-5H2 and BV711, clone Mab11), and respective isotype control mAbs were purchased from BD Biosciences (CA, USA) and Biolegend (CA, USA). Fixable viability dyes used were either eFluor 780 (eBioscience, CA, USA), Zombie Green (Biolegend, CA, USA) or Zombie Aqua (Biolegend, CA, USA) depending on the staining scheme. To detect intracellular cytokines in cultured cells, the latter were stimulated with PMA/Ionomycin/Brefeldin A for the last 5 hs of culture. Then, they were incubated with Cytofix/Cytoperm (BD PharMingen, CA, USA) for 20 minutes (min) in the dark and washed with Perm/Wash solution (BD PharMingen, CA, USA). Following permeabilization, the cells were stained with the respective anti-cytokine mAb. Cells were acquired using FACSAria II (BD Biosciences, CA, USA) and analyzed with FlowJo software (Treestar, OR, USA). Single stained controls were used to set compensation parameters. Fluorescence minus one and isotype-matched Ab controls were used to set analysis gates.