Kaluza acquisition and analysis software

Leukocytes from the thymus, spleen, blood and lymph nodes (LN) were isolated following our previously published methods (31 (link)). The protocol for tumor-infiltrating leukocytes isolation was modified from a method for immune cell isolation developed by Blom et al. (32 (link)). Briefly, tumor tissue was rinsed with ice-cold PBS and crushed with a plastic syringe plunger to pass through a stainless wire mesh strainer. Cells were suspended in 50 ml of PBS. The cell suspension was centrifuged at 60 g for 1 min. The supernatant was transferred into a fresh tube and centrifuged at 480 g for 10 min. The cell pellet was re-suspended in 10 ml of 37.5% Percoll in PBS and centrifuged at 850 g for 30 min. The cell pellet was re-suspended in 5 ml of RBC lysis buffer for 5 min. Cells were washed with PBS+0.1% BSA and collected by centrifugation. Cell phenotype was analyzed by flow cytometry following our previously published methods (33 (link)). Fluorescence Minus One (FMO) controls, including isotype controls, were used to set the gate on the interested cells. For tumor samples, anti-mouse CD45 was used to gate on leukocytes for further analysis. Beckman Coulter Gallios Flow Cytometer and Kaluza acquisition and analysis software (Beckman Coulter, Inc. Miami, FL.) were used to collect and analyze the cell phenotype data.

Lymphocytes were activated by 50 ng/ml PMA and 500 ng/ml ionomycin in RPMI 1640 medium supplemented with 10% FBS and 5 ug/ml Brefeldin A at 37°C for 4 h. Cytokine-producing cells were determined by flow cytometry-based intracellular staining following our previously published method (30 (link)). FMO gate strategy, Beckman Coulter Gallios Flow Cytometer, and Kaluza acquisition and analysis software (Beckman Coulter, Inc. Miami, FL.) were used to collect and analyze the cell phenotype data.