Bv421 conjugated anti human cd8

The peptide-HLA tetramers were prepared as previously described^{32 (link)}. Briefly, ATIGTAMYK (EBV Rta134–142) peptide was refolded with HLA-A*11:01 heavy chain and β2-microglobulin in refolding buffer for 72 h. Refolded complex was subsequently dialyzed against 10 mM Tris (pH 8.0) at 4 °C overnight. Following purification via anion exchange chromatography using HiPrep DEAE 16/10 column (GE Healthcare) equilibrated with 10 mM Tris-HCl (pH 8.0) and gel filtration with a HiLoad 16/60 Superdex 75 preparatory-grade GF column (GE Healthcare), the pHLA monomeric complexes were biotinylated by recombinant BirA enzymes. Assembly of tetrameric pHLA complexes was carried out by the stepwise addition of streptavidin-phycoerythrin (PE) (Invitrogen) or streptavidin-allophycocyanin (APC) (BioLegend) at a molar ratio of 4:1. Cells from the 14-day cultures were first harvested and then washed with PBS. Subsequently, they were stained with 12 µg/ml PE-conjugated pHLA tetramer for 20 min, and BV421-conjugated anti-human CD8 (BD Biosciences) for 15 min. Cells were washed again with PBS before analysis with LSR II flow cytometer (BD Biosciences). Data analyses were performed using FlowJo (Tree Star Incorporated).

Cells from the 14-day cultures were harvested, washed with PBS and stained with 12 µg/ml PE-conjugated pHLA tetramer for 20 min, followed by incubation with BV421-conjugated anti-human CD8 (BD Biosciences) for 15 min. Cells were washed with PBS and analysed with LSR II flow cytometer (BD Biosciences). An aliquot of cells from the 14-day cultures was stained with propidium iodide to determine viability and gating parameters. These gating values were utilised for cells stained with PE-conjugated tetramers and BV421-conjugated anti-human CD8 antibodies. Data analyses were performed using FlowJo (Tree Star Incorporated). TYGPVFMCL and YLLEMLWRL peptides served as controls.