Flow cytometric analysis of tumor infiltrating lymphocytes

Three weeks after the initiation of treatment with a vehicle control or ONC201, the mice bearing E0771 tumors were sacrificed. Their tumors were removed and were crushed and filtered. Dissociated cells were collected and incubated with red blood cell lysis buffer. Afterwards, cells were washed with FACS buffer and incubated for 10 minutes on ice with a mouse CD16/CD32 specific antibody to block Fc receptors (eBioscience, cat # 14-0161-81). Cells were then incubated with primary antibodies from eBioscience for 30 minutes on ice in the dark (CD45: cat # 63-0451-82, CD19: cat # 48-0193-82, CD3: cat # 47-0032-82, NK1.1: cat # 17-5941-82). Cells were washed and resuspended in FACS buffer with 1 μg/mL propidium iodide added for dead cell exclusion. The cells were analyzed using an LSR II flow cytometer (BD Biosciences), and FlowJo software was used to perform data analysis.