70μm nylon cell strainer

Spleens, tibias, and femurs were isolated from mice after incubation, and single cells were harvested with a 70μm nylon cell strainer (VWR) after the residual erythrocytes had been lysed with 1X RBC lysis buffer (BioLegend). Solid tumors were chopped into small pieces and passed through 100-μm nylon cell strainers (VWR), which were then harvested and centrifuged at 13,500 rpm for 15 min at 4 °C. The viability and number of isolated cells from individual tissues were determined using trypan blue exclusion counts.

After mice were anesthetized, blood was collected via central vein, Ammonium-Chloride-Potassium (ACK) lysis buffer was used to lyse red blood cells (RBCs), and then wash with phosphate-buffered saline (PBS). During dissection, spleens were removed and collected in RPMI-1640 solution (11875093, Thermo Fisher Scientific, MA, USA) on the ice. Single cells were obtained after mashing the spleen through a 70-μm nylon cell strainer (VWR International, PA, USA) followed by 10 min treatment with 5 ml RBC lysis buffer (420302, BioLegend, CA, USA) at room temperature. After washing with RPMI-1640 solution, cells were resuspended in RPMI-1640 + 2% FBS. Livers were collected and digested in 1 mg/ml collagenase II for 30 min in 37°C followed by being mashed through a 70-μm strainer to obtain the cell suspension containing hepatocytes and non-parenchymal cells (NPCs). The hepatocytes were removed by centrifugation at a speed of 50 Relative Centrifugal Force (RCF) for 5 min and then the NPCs were collected from the supernatant above after centrifugation at 400 rcf for 5 min. After 10 min of RBC lysis buffer, NPCs were suspended in RPMI-1640 solution.