Anti cd8 af700 clone rpa t8

Fluorochrome-labelled peptide-HLA-A2 tetramers were assembled from monomers from NIH Tetramer Core Facility (Atlanta, GA, USA) and streptavidin Qdots (Thermo Fisher Scientific, Waltham, MA, USA). Tetramer staining was performed as described previously [19 (link)]. Briefly, thawed PBMCs were treated with 50 nmol/l dasatinib for 15 min at 37°C, then pelleted and resuspended in tetramer Qdot master mix for 15 min at 37°C (details in ESM Table 2). Cells were incubated with LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific) for 10 min at room temperature and stained for 30 min at 4°C with titrated concentrations of the following antibodies: anti-CD8–AF700 (clone RPA-T8) (BD Biosciences, San Jose, CA, USA); anti-CD4–FITC (clone OKT4); anti-CD14–FITC (clone 61D3); anti-CD16–FITC (clone eBioCB16); anti-CD20–FITC (clone 2H7); and anti-CD40–FITC (clone 5C3) (dump channel; eBioscience, San Diego, CA, USA). Data were acquired using a modified FACSAria II flow cytometer (BD Biosciences). All flow cytometry data were analysed with FlowJo software version 10 (Tree Star, Ashland, OR, USA).

Flow cytometry was used to analyze the phenotypes of freshly isolated cells. T cell phenotype was determined using the following fluorochrome-conjugated monoclonal antibodies from BD Biosciences, San Jose, CA: anti-CD4 FITC (clone RPA-T4, RRID: AB_2562052), and anti-CD8AF700 (clone RPA-T8, RRID: AB_396953) (15 (link)). The samples were exposed to monoclonal antibodies for 15 min at room temperature in the dark, washed, and events were monitored using a Beckman Coulter Navios instrument (Beckman, San Jose, CA, USA). T helper and cytotoxic lymphocytes were gated by positive surface staining for CD4 and CD8 and were expressed as a percentage of gated lymphocytes (16 (link)).