Cd8 t cell enrichment cocktails

Ex vivo-purified primary CD4 and CD8 T cells were obtained from the blood of a healthy donor using Rosettesep CD4 and CD8 T cell enrichment cocktails, respectively (Stemcell Technologies). Primary T lymphocytes were maintained in RPMI 1640 supplemented with 10% FBS and 30 U of rIL-2/ml. T cells were expanded using Dynabeads human T-activator CD3/CD28 (Life Technologies) immediately after isolation. The purity and activation status of CD4 and CD8 T cells were determined by using flow cytometry and antibodies against CD4 (BD Biosciences), CD8 (BioLegend, clone RPA-T8), CD3 (BD Pharmingen), and CD25 (BioLegend, clone BC96). The resting cell population was removed from T-cell receptor (TCR) stimulation 24 h prior to the experiment, and the activation status was determined at the time of plating for the viability test. Viability was determined using the fixable viability dye eFlour 660 (eBioscience) at 24 hpi with VSV-XN2 or VSV-gp160G at the indicated MOI. Activated primary CD4 T cells were also infected with wild-type VSV Indiana serotype at an MOI of 0.01 at 24 hpi and measured for viability compared to VSV-XN2 and VSV-gp160G. Prior to analysis, cells were fixed in 2% paraformaldehyde. Cells were analyzed using LSR-Fortessa-HTS.