Live dead fixable dye

Single-cell preparations were stained with antibodies from eBioscience, BD Biosciences, and Biolegend. Cytokines, Foxp3, Ki67, and Bcl-2 were stained using the eBioscience Fix/Perm kit. Cytokines were also stained in buffer containing 0.5% Saponin (Sigma). Dead cells were excluded by use of a Live/Dead fixable dye (Biolegend). Samples were acquired using a Fortessa (BD Biosciences) and analyzed with FlowJo software (Treestar). Cell sorting was performed using an Influx (BD Biosciences).

Single-cell suspensions of spleens were prepared by mechanical disruption. For cell isolation from livers, mechanical disruption was followed by digestion with Collagenase D (0.1 U/ml, Roche) for 30 min at 37°C. Then, lymphocytes were isolated using Histopaque-1083 and high-density centrifugation (400 g at 20°C for 20 min). When indicated, ex vivo-isolated lymphocytes and in vitro-differentiated CD8⁺ T cells were stained with antibodies against CD8 (53–6.7), CD45.1 (A20), CD62L (MEL-14), CD44 (IM7), KLRG1 (2F1), IL-18R (BG/IL18RA), CXCR3 (CXCR3-173), CD127 (A7R34), and PD-1 (J34). For flow-cytometric detection of cell surface ST2, splenocytes were stained with digoxigenin-coupled anti-mouse ST2 antibody (DJ8). For detection, a PE-coupled anti-digoxigenin Fab antibody (Roche) was used. To augment the PE signal, we performed two rounds of amplification using the PE FASER Kit (Miltenyi Biotec). LCMV-specific CD8 T cell response to the dominant glycoprotein-derived epitope GP33 was assessed by MHC class I tetramer staining as described previously (16 (link)). Samples were acquired on a FACS Canto II (BD), and analyzed with FlowJo (BD). Dead cells and doublets were excluded by a combination of forward scatter height and width gating and the usage of propidium iodide or a LIVE/DEAD fixable dye (BioLegend).