Fitc anti human hla a2 antibody

T2A2 cells are TAP-deficient T2 cells expressing HLA-A2 molecules on the cell surface.^{21 (link)} Cells were maintained in IMDM (Cat#SH30228.01, HyClone, Waltham, Massachusetts, USA) with 20% FBS (Cat#S711-001S, Lonsera, Uruguay) and 1% penicillin-streptomycin (Cat#SV30010, HyClone, Waltham, Massachusetts, USA). PBMCs were isolated from whole blood of healthy donors by density gradient centrifugation using Ficoll-Paque PLUS (Cat#17144003, GE, Chicago, Illinois, USA). Briefly, the blood samples were diluted by adding PBS buffer at a ratio of 1:1, gently mixed upside down. A total of 10 ml Ficoll separation solution was slowly added into the centrifuge tube, and then centrifuged for 20 min. The PBMCs were saved and washed with PBS. PBMCs were stained with FITC anti-human HLA-A2 antibody (Cat#343303, Biolegend, San Diego, California, USA) at 4°C for 30 min in the dark, and were acquired on flow cytometer FACS Canto (BD, Franklin Lakes, New Jersey, USA). Aliquots of HLA-A2⁺ PBMCs were cryopreserved at 90% FBS (Cat#S711-001S, Lonsera, Uruguay) and 10% DMSO (Cat#D2650, Sigma-Aldrich, Burlington, USA) and stored in liquid nitrogen or at −80°C until usage.

PBMCs were isolated from peripheral venous blood of healthy donors, convalescent COVID-19 patients and SARS-CoV-2 vaccinees. The HLA-A2⁺ donors were identified by using flow cytometry. Briefly, 10⁶ PBMCs were stained with FITC anti-human HLA-A2 antibody (BioLegend) at 4°C in the dark for 30 min, and acquired by using flow cytometer. HLA-A2 positive PBMCs samples were further stained with PE labeled tetramer (home-made), PerCP labeled human CD8⁺ antibody (BioLegend), APC labeled human CCR7 antibody (BioLegend), FITC labeled human CD45RA antibody (BioLegend) and acquired with flow cytometer FACS Canto (BD).

Antibody were purchased from BioLegend, including FITC anti-human HLA-A2 antibody (Cat#343303), PE anti-human HLA-A2 antibody (Cat#343305), anti-human CD28 antibodies (Cat#302901), APC anti-human-CD69 (Cat#310909), APC anti-human CD8 (Cat#344721), PerCP anti-human IFN-γ (Cat#502524), and FITC anti-human granzyme B (Cat#515403). Flex-T monomer (Cat#280003) and PE conjugated streptavidin (Cat#405203) were purchased from BioLegend. Ficoll-Paque PLUS (Cat#17144003) was from GE and DMSO (Cat#D2650) was from Sigma-Aldrich. IMDM (Cat#SH30228.01) and penicillin-streptomycin (Cat#SV30010) were from HyClone, and FBS (Cat#S711-001S) was purchased from LONSERA. PBS (Cat#C10010500BT) was from Gibco (Waltham, Massachusetts, USA). D-biotin (Cat#2110450) was purchased from Invitrogen (Waltham, Massachusetts, USA), and Mitomycin C (Cat#50-07-7) was from Sinochem Holdings (Beijing, China). CFSE (Cat#T6802) was from Targetmol (Boston, Massachusetts, USA). EasySep Human CD8⁺ T Cell Isolation Kit (Cat#17953) was from Stem Cell Technologies (Vancouver, British Columbia, Canada), and IL-2 (recombinant human interleukin-2(¹²⁵Ala) injection) was from SL PHARM (Beijing, China). Leuko act cktl with GolgiPlug (Cat#550583) was purchased from BD.