Anti cd95 pe cy5

For assessment of cytokines and p65 in T-cell subsets by flow cytometry, monoclonal antibodies and respective isotype controls were titrated to determine optimal staining concentration (Supplementary Table 1). Cells were centrifuged, supernatant discarded, and resuspended in Fluorescence Activated Cell Sorting (FACS) buffer (PBS and 0.1% BSA) containing the appropriate cell surface antibody or isotype control using BD Pharmingen Abs: anti-CD4/8 FITC or anti-CD95 PE-Cy5. Cells were incubated at 4 °C in the dark, for 30 min, and washed with FACS buffer afterwards. To preserve staining, the cells were fixed using FACS fix solution (PBS containing 0.1% BSA and 1% PFA). Following fixation, cells were washed with FACS buffer, and resuspended in FACS permeabilisation buffer (PBS containing 0.1% BSA and 0.1% saponin). Following permeabilisation, cells were washed and resuspended in FACS permeabilisation buffer, where appropriate antibodies for intracellular proteins were added to the cells: anti-Tbet/GATA3/RORγt/FOXP3 Alexa Fluor 647; anti-IL-2/IFN-γ/IL-4/IL-17/TGF-β PE (all from BD Pharmingen); or anti-NF-κBp65 PE (BioLegend). Cells were incubated in the dark for 45 min at 4 °C, and subsequently washed with FACS perm buffer, and resulting pellets were resuspended in FACS buffer for analysis by flow cytometry.