Analyzing Lymphocyte Responses in LLC Tumor Model

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).