Anti tnfα percp cy5.5 clone mab11

For activation marker and cytokine analysis, 1x10⁶ human GUCY2C-directed CAR-transduced human T cells were stimulated for 6 hours in tissue culture plates coated overnight at 4°C with 10 μg/mL human GUCY2C or BSA control antigen in PBS or with 1X Cell Stimulation Cocktail (PMA/Ionomycin, eBioscience) added at the time of plating CAR-T cells. All conditions included 1X Protein Transport Inhibitor Cocktail (eBioscience) at the beginning of the incubation period. Cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Invitrogen) in PBS for 10 minutes and subsequently stained for surface markers using anti-CD8–Qdot 800 (clone 3B5, Invitrogen) and anti-CD69–APC (clone L78, BD Biosciences) in PBS 0.5% BSA for 25 minutes. Intracellular cytokine staining was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences) consisting of fixation with Cytofix/Cytoperm buffer for 20 minutes and staining with anti-GFP–Alexa Fluor 488 (Invitrogen), anti-IFNγ–BV605 (clone 4S.B3; BioLegend), anti-TNFα–PerCP-Cy5.5 (clone Mab11; BD Biosciences), and anti-IL2–PE (clone MQ1-17H12; BD Biosciences) in BD perm wash buffer for 45 minutes. Cells were fixed in 2% PFA and analyzed on a BD LSR II flow cytometer. Analyses were performed using FlowJo v10 software (Tree Star).