Ack lysing buffer

LLC cells were seeded in 12-well plates at a density of 1×10⁵ cells/well for 24 h and were transfected with IDO1- or GL2-siRNA. After 24 h of transfection, lymphocytes isolated from the spleen of LLC-bearing C57BL/6 mice (33 (link)). Briefly, spleens (~1 cm in length and 30 mm in width) were placed on a 40 µm Falcon Cell Strainer (VWR International, LLC.) and gently squashed with a plunger. The cell suspension was collected to centrifuge at 4°C, 250 g for 5 min, then further isolated using ACK Lysing Buffer (Beijing Solarbio Science & Technology Co., Ltd.) to lyse red blood cells. Lymphocytes were added to the LLC cells at a density of 5×10⁵ cells/well and were cultured at 37°C with 5% CO₂ for 48 h. Lymphocytes were subsequently collected and stained with anti-CD4-FITC (cat. no. 553047; 1:200; BD Pharmingen; BD Biosciences), anti-CD8-PE (cat. no. 12-0081-82; 1:200; eBioscience; Thermo Fisher Scientific, Inc.), anti-PD-1-APC (cat. no. 17-9985-82; 1:200; eBioscience; Thermo Fisher Scientific, Inc.) and anti-BTLA-PE (Cat. no. 12-5950-82; 1:200; eBioscience; Thermo Fisher Scientific, Inc.), incubated at 4°C in dark for 30 min, then detected using flow cytometry (BD FACSCanto II; BD Biosciences). The data were analyzed suing FlowJo version 10 software (Becton, Dickinson and Company).

Blood samples (100 μL) were collected from each group on day 30 after inoculation of tumor cells, and the red blood cells were then lysed by ACK lysing buffer (Solarbio). To detect DCs, cells were stained with FITC‐labeled anti‐mCD11c and PE‐labeled anti‐mCD80 antibody for 30 minutes. To qualify the CD4⁺/CD8⁺ T populations, cells were stained with FITC‐labeled anti‐mCD4 or FITC‐labeled anti‐mCD8 antibody for 30 minutes. To detect the Treg populations in the blood of each experimental group, the cells were incubated with FITC‐labeled anti‐mCD4 for 30 minutes, then the cells were stained with PerCP‐Cyanine 5.5‐labeled anti‐mFoxP3 antibodies for 30 minutes after fixed and permeabilized using the Foxp3 staining kit (eBioscience). T‐cell subsets were measured by flow cytometry (Becton Dickinson).