Rbc lysis buffer 10

The peritoneal cavity was opened to expose the liver, and 5 to 10 ml of 1× PBS were injected into the hepatic portal vein using a 27G needle to perfuse the liver. The gall bladder was then removed, and all lobes of the liver were collected and mashed through a 70-μm filter sitting in a 50-ml tube using a syringe plunger. The filter was rinsed with 1× PBS/2% FCS/5 mM EDTA three to four times during the mashing procedure. The cell suspension was centrifuged at 300g for 5 min at 4°C and then supernatant-aspirated, and the cell pellet washed with 10 ml of 1× PBS/2% FCS/5 mM EDTA. Following centrifugation and aspiration of the supernatant, the pellet was resuspended in 25 ml of isotonic Percoll [8.44 ml of Percoll (Healthcare Biosciences, Uppsala, Sweden; catalog no. 17-0891-01)/0.47 ml of 20× PBS/16.09 ml of 1× PBS] at room temperature (RT). Liver cell suspensions were centrifuged at 693g for 12 min at RT with no brake; the leukocyte pellet at the bottom of the tube was then washed with 10 ml of 1× PBS/2% FCS/5 mM EDTA and RBC-lysed (RBC lysis buffer 10×, BioLegend, catalog no. 420301). Following washing with 1× PBS/2% FCS/5 mM EDTA, the pellet was resuspended in 2.4G2 blocking solution before flow cytometry staining.