100mm cell strainers

Tumors or livers were finely chopped and processed on a gentleMACS^™ Octo Dissociator using the pre-set 37C_m_TDK_2 or 37C_m_LDK_1 program. Following dissociation, tumors were quenched with 5ml of RPMI media supplemented with 10% FBS, 1% penicillin/streptomycin and 0.5% L-glutamine, and passed through 100mm cell strainers (Falcon, cat. #08-771-19) to further separate cells. An additional 5ml wash was performed on the C-tube and passed through the cell strainer, followed by a 5ml wash over the cell strainer to flush any remaining cells. The flow through was spun and ACK lysis (Quality Biological, cat. #118-156-101) was performed with 4mls of ACK lysis buffer followed by a 4-minute incubation on ice. Cells were quenched with RPMI complete media and spun down. For the formation of a Percoll gradient, a stock Percoll solution was prepared by diluting the supplied Percoll (GE Healthcare Life Sciences, cat. #17-0891-01) with 10X PBS. This was further diluted with 1X PBS to form 80% and 40% Percoll solutions. Cell pellets were resuspended in 40% Percoll and underlayed with 80% Percoll and then spun at 3200 rpm (no brake) for 25 minutes at room temperature. The immune cell layer was then removed and 1×10⁶ cells were plated per well in a 96-well plate for flow cytometry analysis.