Streptavidin allophycocyanin apc

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).

Unless stated, otherwise all reagents used were ice cold and samples were kept on ice at all time. 1 × 10⁶ A549 or Calu3 cells were blocked with 5% BSA, PBS on ice. After 30 min, cells were stained with either 50 μg/mL biotin conjugated Sambucus nigra (Elderberry Bark) -SNA-I (EY Lab, San Mateo, USA), or 50 μg/mL biotin conjugated Maackia amurensis Lectin –MAA (EY Lab, San Mateo, USA) or 0.25 μg of Alexa Fluor 488 mouse anti-human EGFR antibody, clone AY13 (Biolegend, San Diego United States), or 0.25 μg Alexa Fluor 488 mouse Ig gamma −1 (IgG1), clone MOPC-21 (Biolegend, San Diego United States) in 50 μL ice cold 1% BSA in PBS. Cells were incubated on ice for 1 h and washed three times with 2% BSA in PBS. After washing, 4 μg/mL allophycocyanin (APC) streptavidin (Biolegend, San Diego United States) were added to sample stained with biotin-MAA and biotin-SNA I. After 30 min of staining, cells were washed twice with 2% BSA in PBS and resuspended in 400 μL of 2% BSA in PBS containing 0.3 μg/mL propidium iodide (PI) for flow cytometry analysis. All samples were analysed with BD LSRFortessa^TM Flow Cytometer (Becton, Dickinson and Company, Frankin Lakes, United States). All data were analzed with Flowjo software, version 10 (Becton, Dickinson and Company, Frankin Lakes, United States).