Cd8b fitc

Peripheral blood samples were collected from the tail vein of the mice (21-day drug treatments) into Microtainer blood collection tubes (BD) after which the samples were stained with CD45.1-APC-Cy7 (Biolegend, clone 30-F11, cat. 103116), CD3-APC (Biolegend, clone 17A2, cat.100236), CD4-V500 (BD, clone RM4–5, cat. 560782), CD8b-FITC (eBioscience, clone eBio H35–17.2, cat. 11–0083–85), NK1.1-PE (BD Biosciences, clone PK136 cat. 557391) and red blood cells were lysed after with FACS lysing solution (BD). The samples were acquired with FACSVerse (BD Biosciences) and analysed with FlowJo version 10.3 (TreeStar).

After red blood cell lysis, stainings were performed in the presence of 10 μl/mL of FcR blocking reagent (Miltenyi) in PBS with 2% FBS. For flow cytometry analysis, acquisition and data analysis were conducted on a LSR Fortessa cytometer (BD Biosciences) and FlowJo v10.7 software. For Next Generation Sequencing, cells were sorted using Influx Cell Sorter (BD Biosciences) and BD FACSTM v1.2.0.142 software (BD Biosciences). We used the following antibodies from BD Biosciences or eBiosciences: CD11b-PE-Cy7, CD19-PE-Texas Red, CD25-APC, CD253-APC, CD274-PE, CD3e-PE-Cy7, CD4-efluor 450, CD45-BV711, CD8b-FITC, F4/80-Alexa-eFluor 647, FoxP3-PE, Granzyme B-PE, Ly6G-FITC, MHC Class II-APC efluor 780, NK1.1-FITC, PD1-APC. Live and dead cells were distinguished using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life technologies). The gating strategy is shown in Fig. S1.