Streptavidin phycoerythrin pe

The extracted DNA were amplified by nested PCR reactions using PGMY09/PGMY11 and GP5+/GP6+ primers, as previously described by Zubach et al. [27 (link)] and Awua et al. [26 (link)]. The typing of 46 mucosal HPV types were carried out by a multiplex system based on the xMAP® technology, as previously described by Zubach et al. [27 (link)] and reported by Awua et al. [26 (link)]. Briefly, 46 fluorescence sortable microspheres (Luminex Corporation, Austin, TX) were coupled to the 46 specific probes for the HPV types; 6, 11, 13, 16, 18, 26, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 89, 90, and 91. The double-stranded second-round PCR products, labelled with biotin, were made single-stranded by digestion with 2 µL of bacteriophage T7 gene6 exonuclease (New England BioLabs, Pickering, ON, Canada) that removed the non-labelled strand after 40 minutes incubation at room temperature. The single-stranded HPV DNA were incubated for hybridization at 60°C for 10 minutes. Streptavidin-phycoerythrin (PE) (Invitrogen) in 1-tetramethyl ammonium chloride (TMAC) (Sigma), was subsequently added and incubated for 5 minutes at 60°C. Genotype specific hybridizations were detected on a Luminex Liquid Chip 200 flow cytometer (Qiagen) using the Luminex IS software (Luminex).

HLA-peptide tetramers were produced as previously described^{46 (link)}. Briefly, to form stable pHLA complexes, peptides SSCSSCPLSK (EBV LMP2_340–349), SSSPQCPLSK (hybrid of human p53_313–317 and EBV LMP2_345–349), KICMQCPLSK (hybrid of human ribosomal protein S4_63–67 and EBV LMP2_345–349) and KTYGECPLSK (hybrid of human NADH dehydrogenase subunit C2_106–110 and EBV LMP2_345–349) were refolded individually with HLA heavy chain and β2 m in refolding buffer for 72 h. Refolded products were then dialyzed against 10 mM Tris-HCl (pH 8.0) at 4 °C overnight. Dialyzed complexes were purified by anion exchange chromatography using HiPrep DEAE 16/10 column (GE Healthcare) equilibrated with 10 mM Tris-HCl (pH 8.0), followed by gel filtration with a HiLoad 16/60 Superdex 75 preparatory-grade GF column (GE Healthcare). The purified, refolded pHLA monomeric complexes were then biotinylated by recombinant BirA enzymes. Tetrameric pHLA complexes were assembled by the stepwise addition of streptavidin-phycoerythrin (PE) (Invitrogen) or streptavidin-allophycocyanin (APC) (BioLegend) at a molar ratio of 4:1.

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).