Fixative free lysing solution

Blood was collected weekly to assess T cell phenotype and absolute count. On day 42 prior to organ harvest, mice were intravenously injected with 1.5µg of anti-CD4-BV650 and anti-CD8a-BV650 antibody 2 minutes before euthanasia to label circulating blood cells. The spleen, axillary lymph nodes, bone marrow, kidneys, liver, lungs, and blood were then procured. Kidney and lung samples were chopped and digested for 30 minutes at 37˚C with 2mg/ml Collagenase (type 4, Sigma-Aldrich) and 50µg/ml DNAse (ThermoFisher) in HBSS. Digested lungs were then homogenized, filtered, and washed in FACS buffer (PBS with 2% FBS). Livers were homogenized manually, filtered through 40μm strainers, and spun lightly at 300rpm to pellet the hepatocytes. The liver supernatant and digested kidneys were resuspended in a 40% Percoll solution, overlaid on 70% Percoll, and spun at 2000rpm for 20 minutes with the brake off. The buffy coats were isolated and washed in FACS buffer. Bone marrow from tibia and fibula were flushed with a 21-gauge needle using PBS and homogenized into single cell suspensions. Spleens and lymph nodes were processed into single cell suspensions, and blood, spleen, and bone marrow were lysed with Fixative-Free Lysing Solution following manufacturer’s instructions (Invitrogen). Each tissue was then washed in FACS buffer and stained with antibodies for flow cytometry.

The tumors xenografts were harvested and cut into small piece under sterile conditions, and treated with dispase (1 mg/ml) and collagenase type IV (1 mg/ml), and incubated at 37C for 20 min, then the tumor tissue was homogenized with a serological pipette, and supernatants were collected. The remaining tissue was treated with the same solution for an additional 2–4 times until almost all tissue was digested. The supernatants were spun at 300 g for 5 min, the cell pellet was re-suspended with MEM medium after discarding the supernatant and was treated with fixative-free lysing solution (www.invitrogen.com) for 10 min in the dark to destroy the blood cells, then filtered with a 100 um cell strainer and spun again. Finally the cell pellet were re-suspended, and re-cultured with MEM (parental SK cell culture condition).