Fluorescein isothiocyanate anti human cd4

T cells were left untreated, harvested after 48 hours of activation with anti-CD3/CD28 Dynabeads, or collected at the end of phase 1 and phase 2 of co-culture, washed once with PBS, and stained with the Zombie Aqua Fixable Viability Kit (Biolegend), followed by antibody stain: PE/Cy7 anti-human CD69, Brilliant Violet 711 anti-human CD25, fluorescein isothiocyanate anti-human CD4, Alexa Fluor 700 anti-human CD8a, PerCP/cyanine 5.5 anti-human CD3, APC anti-human/mouse integrin β7 (all from Biolegend, San Diego, CA). Cells were fixed in 1% paraformaldehyde and analyzed using an LSRFortessa (Becton, Dickinson and Company, Franklin Lakes, NJ) in FACS buffer (0.1% bovine serum albumin, 0.05% sodium azide in PBS). Flow cytometry samples were gated on singlets via standard saline citrate, and then lymphocytes using FSC and standard saline citrate. Gates were drawn to exclude CD14+ CD19+ and dead cells, and expression levels and the percentage of positive cells were quantified using FlowJo v10.4 software (BD).