Cd11b af700 m1 70

Cells were surface stained using CD8-efluor 450 (53–6.7; 0.25 µg/test; eBioscience, San Diego, CA, USA), CD45-BV605 (30-F11; 0.5 µg/test; Biolegend), CD11b-AF700 (M1/70; 0.25 µg/test; eBioscience), and MHC-1 APC (AF6-88.5.5.3; 0.5 µg/test; eBioscience) prior to permeabilization and fixation using the Cytofix/cytoperm kit (eBioscience). Cells were then stained for IFN-γ-BV650 (XMG1.2; 0.25 µg/test; Biolegend), TNF-BV711 (MP6-XT22; 0.5 µg/test; Biolegend), Granzyme B-AF647 (GB11; 5µL/test; Biolegend), Ki67-PE-Cy7 (SolA15; 0.125 µg/test; eBioscience), and IL-6-PerCP-eFluor710 (MP5-20F3; 0.125 µg/test; eBioscience) according to the manufacturer’s protocol.

Aliquots of up to 3 × 10⁶ cells were blocked using an anti-CD16/CD32 blocking antibody (BioLegend, San Diego, CA) and labelled with fluorescently conjugated antibodies in the dark as described previously (Thomson et al. 2018). The following antibodies were purchased from BioLegend: CD4-AF488 (GK1.5), CD8α-PE/Cy7 (53-6.7), CD11b-PE (M1/70), CD44-BV421 (IM7), CD45-BV510 (30-F11), CD45-PE/Cy7 (30-F11), CD62L-PE (MEL-14), CD64-APC (X54-5/7.1), F4/80-FITC (BM8), Ly6G-AF700 (1A8), MHCII-BV510 (M5/114.15.2), NK1.1-BV605 (PK136) and TCRβ-PerCP/Cy5.5 (H57-597). The following were purchased from eBioscience (San Diego, CA): CD11b-AF700 (M1/70) and Ly6C-eFluor 450 (HK1.4). Dead cells were excluded from analysis using Fixable Viability Dye eFluor 780 (eBioscience, San Diego, CA). Cells were analysed using an LSRII (BD Biosciences, San Jose, CA). Data were analysed using FlowJo v10 (Tree Star, Ashland, OR).