Zombie nir fixable viability dye

Isolated PBMCs (2 × 10⁶) were centrifuged and resuspended in 75 μl FACS buffer (phosphate-buffered saline (PBS) and 2% fetal bovine serum (FBS)) and 5 μl Fc receptor block (BioLegend, no. 422302) for 5 min at room temperature. For samples stained with anti-IgG, it was observed that Fc block inappropriately interfered with staining, so a preincubation step of the anti-IgG alone for 5 min at 22 °C was added before the addition of the block. Next, 25 μl of antibody cocktail (Supplementary Table 3) was added (100 μl staining reaction), and samples were incubated for 20 min at 4 °C. Cells were washed in PBS and resuspended in a PBS dilution of Zombie NIR fixable viability dye (BioLegend, no. 423106). Cells were washed and fixed in 0.8% paraformaldehyde for 10 min at 22 °C in the dark before a final wash and resuspension for analysis.
Cells were analysed on a Cytek Aurora flow cytometer using Cytek SpectroFlo software. Up to 3 × 10⁶ cells were analysed using FlowJo v.10 (Treestar) software.

Cells were stained with Zombie NIR Fixable Viability dye (catalog no. 423106; BioLegend, San Diego, CA, United States), which is non-permeant to live cells but is permeant to cells with compromised membranes, i.e., dead cells. The cells were then pre-incubated with purified anti-mouse CD16/32 antibody (catalog no. 101310; BioLegend) to block non-specific immunoglobulin binding to the Fc receptors. Finally, the cells were incubated with specific antibodies or isotype controls according to the manufacturers’ guidelines. The antibodies used were: Pacific Blue anti-mouse CD45; allophycocyanin-conjugated (APC) anti-mouse CD3; fluorescein isothiocyanate–conjugated (FITC) anti-mouse CD8a; peridinin chlorophyll protein complex–conjugated (PerCP) anti-mouse CD4; FITC anti-mouse CD19; phycoerythrin-conjugated (PE) anti-mouse CD366; Pacific Blue Rat immunoglobulin (Ig)G2b, κ Isotype Ctrl; APC Rat IgG2b, κ Isotype Ctrl; FITC Rat IgG2a, κ Isotype Ctrl; PerCP Rat IgG2b, κ Isotype Ctrl; and PE Rat IgG2a, κ Isotype Ctrl (all from BioLegend). The cells were detected and analyzed using a FACSAria III flow cytometer (BD Biosciences, San Jose, CA, United States); positive cells were defined using an isotype control or a fluorescence minus one control.

Stimulated cells were washed with PBS and stained for 20 min at room temperature with Zombie NIR? Fixable Viability Dye (BioLegend). Cells were surface stained for 30 min at room temperature with anti-CD3 Brilliant Violet 711 (UCHT1; BD Horizon), anti-CD4 Brilliant Violet 570 (RPA-T4; BioLegend), anti-CD8 PerCP-Cy5.5 (SK1; BioLegend), anti-PD-1 PE (EH12.2H7; BioLegend), and anti-BTLA APC (MIH26; BioLegend). Cells were washed with PBS containing 1% FCS and fixed with FACS Lysing Solution (BD Biosciences), then washed with Perm/Wash Buffer (BD Biosciences). Cells were stained intracellularly for 30 min at room temperature with anti-IFN-γ FITC (B27; BD Pharmingen), anti-TNF-α Brilliant Violet 421 (Mab11; BioLegend), and anti-CD152 PE-CF594 (BNI3; BD Horizon), washed with Perm/Wash Buffer (BD Biosciences) and suspended in PBS prior to acquisition.