Anti cd45.1 fitc clone a20

Blood samples from mice were depleted of red blood cells by washing with ACK lysis buffer. They were then stained with the following fluorophore-conjugated antibodies: anti-CD8 APC (clone 53-6.7, #100712; BioLegend) and anti-CD45.1 FITC (clone A20, #110706; BioLegend). For the day 18 timepoint after tumor challenge, a larger antibody panel was used: anti-CD8 PacificBlue (clone 53-6.7, #100725; BioLegend), anti-CD45.1 FITC (clone A20, #110706; BioLegend), anti-IL7Rα APC (clone A7R34, #135012; BioLegend), and anti-CX3CR1 PE (clone SA011F11, #149006; BioLegend). Antibodies were diluted in a staining buffer consisting of 2% heat-inactivated FBS in PBS, and staining was done at 4°C for 20 min. Samples were then analyzed on a Sony SP6800 Spectral Analyzer. Gating and analysis of flow cytometry data was done in FlowJo 10.8.1. Samples that yielded fewer than 200 CD8 cells were discarded from analyses requiring the estimation of the circulating frequency of CD8⁺ CD45.1⁺ cells.