Mouse treg staining kit 1

TRIzol LS Reagent was purchased from Invitrogen. The mouse Treg staining kit#1 was purchased from eBioscience. DT from corynebacterium diphtheria was purchased from Sigma-Aldrich. The purified rat anti-mouse CD25 antibody was purchased from BD Pharmingen. A peripheral blood lymphocyte isolation kit was purchased from Tianjin Haoyang Biological Company.

Tumor tissues were processed into single-cell suspensions by mincing, as well as chemical (Murine Tumor Dissociation Kit, Miltenyi Biotec) and mechanical (gentleMACS Dissociator) dissociation, per manufacturer recommendations. Suspensions were filtered through a 100 μm filter and washed with 1% BSA in PBS prior to blocking nonspecific staining with anti-CD16/32 (BioLegend) antibody. Cell surface staining was performed using fluorophore-conjugated anti–mouse CD45.2 (clone 104), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD31 (clone 390), PDGFR (clone APA5), PD-L1 (clone 10F.9G2), H2-K^b (clone AF6-88.5), CD107a (clone 1D4B), PD-1 (clone RMP1-30), CD11b (clone M1/70), Ly6G (clone 1A8), Ly6C (clone HK1.4), CTLA-4 (clone 9H10), CD39 (clone Duha59), CD11c (clone N418), F4/80 (clone BM8), and CD206 (clone C068C2) from BioLegend, and IL-12Rb2 (clone 305719) from R&D Systems. FoxP3⁺ Treg staining performed with the mouse Treg Staining Kit #1 (eBioscience) as per manufacturer’s protocol. Cell viability was assessed via staining with Sytox (Thermo Fisher Scientific) or Zombie (BioLegend) dyes. All analyses were performed on a BD Fortessa analyzer running FACSDiva software and interpreted using FlowJo V.X10.0.7r2.