Live dead aqua

For analyzing DR2a and DR2b expression levels on B cells and monocytes, PBMCs from HLA-DR15⁺ donors were incubated with human IgG (Sigma-Aldrich) to block unspecific antibody binding to Fc-receptors, labeled with Live/Dead® Aqua (Invitrogen), and then stained for surface marker using fluorochrome-conjugated antibodies, including Alexa Fluor 488-conjugated anti-DR2a antibody (5 μg/ml, One Lambda, Thermo Fisher Scientific) and Alexa Fluor 647-conjugated anti-DR2b antibody (5 μg/ml, One Lambda, Thermo Fisher Scientific), at 4°C. For analyzing CD69, CD25, and TCR α/β expression on TCCs after stimulation, TCCs were harvested at different time points after stimulating with peptides and washed twice with PBS. TCCs were then incubated with human IgG (Sigma-Aldrich), labeled with Live/Dead® Aqua (Invitrogen), and stained for surface marker using fluorochrome-conjugated antibodies, including anti-human CD69 antibody, anti-human CD25 antibody, anti-human TCR α/β antibody, anti-human CD4 antibody, and anti-human CD3 antibody (Biolegend; Key Resources Table), at 4°C. Cells were washed twice after staining and resuspended with cold PBS containing 2mM ethylenediamine tetraacetic acid (EDTA, AppliChem) and 2% FCS (Eurobio). Measurement was performed using LSR Fortessa Flow Cytometer (BD Biosciences) and data were analyzed using FlowJo (Tree Star).