Anti cd8 apc clone rpa t8

Proliferation assays used cryopreserved PBMC which were thawed, then rested overnight before labelling with Carboxyfluorescein succinimidyl ester (CFSE) as previously described [17 (link)]. Briefly, PBMC were labelled at 5–10 x 10⁶ cells/ml of pre-warmed phosphate buffered saline (PBS)/1μM CFSE at 37°C for 10 minutes, followed by quenching with five volumes of ice cold R10 and two washes. After eight days in culture with 3 μg/ml JEV peptide pools, cells were stained with near-infra-red (IR) viability dye (molecular probes), anti-CD3-AF700 (clone UCHT1), anti-CD4-PE (clone RPA-T4), anti-CD8-APC (clone RPA-T8) and anti-CD38-PE-Cy7 (clone HIT2) fluorescent antibodies (all from BD biosciences) for flow cytometry.

Peripheral blood mononuclear cells were isolated from buffy coats by healthy donors using Lymphoprep (Axis-Shield) and further separated based on CD4 with magnetic beads (CD4+ T-cell isolation kit, Miltenyi Biotec, 130-096-533). The CD4-positive fraction was used as-is, and from the CD4 negative fraction, CD8+ cells were enriched by staining with fluorescently labeled antibodies against CD3+ (Anti-CD3-Facific Blue, clone UCHT1, BioLegend, 300418) and CD8+ (Anti-CD8-APC, clone RPA-T8, BD, 555369) followed by fluorescence-activated cell sorting. Naïve CD4+ T-cells were isolated with the help of a Naïve CD4 T-Cell Isolation Kit (Miltenyi Biotec, 130-096-533).