Rpmi 1640 1 medium

Spleen lymphocytes were prepared as the previous study reported.²⁷ Briefly, the spleen was grinded in the complete RPMI 1 × 1640 medium (Gibco, Palo Alto, USA) with 10% FBS and 1% PS, the red blood cell (RBC) was lysis by erythrocyte lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH7.2), then the suspensions filtered through a 70 μm nylon mesh, and washed by PBS with 1% FBS and counted through a microscope.
The method of isolated hepatic lymphocytes as previously described with some modifications.²⁷, ²⁸ Briefly, after anesthetization, the liver was perfused with 10 mL of sterile PBS through the portal vein, and subsequently excised and cut into small pieces, digested in the buffer contained with collagenase IV 5% (Sigma‐Aldrich) and 100 μg/mL DNase I (Roche Life Science) at 37°C for 30 minutes. Then, the liver suspensions were passed through 70 μm nylon mesh and centrifuged at low speed (60 × g, 2 minutes) to remove hepatocytes. The hepatic lymphocytes were separated using 35% Percoll (Sigma‐ Aldrich) by centrifuging at 600 × g for 20 minutes. Lymphocytes were collected and lysis in erythrocyte lysis buffer to remove RBC.