To prepare splenocytes, spleens were ground in incomplete RPMI 1640 medium (Gibco, Grand Island, NY, USA). Red blood cells (RBCs) were lysed with an RBC lysis solution (Sigma Aldrich). After being washed in staining buffer containing PBS with 1% fetal bovine serum (FBS), cells were filtered through 200-gauge mesh and collected.
Hepatic lymphocytes were prepared as described previously with some modifications [18 (link)]. The remaining hepatic tissues were washed in D-Hank’s solution (Invitrogen, Carlsbad, CA, USA) via the portal vein, cut into pieces and digested in 20 mg Collagenase B (Invitrogen) at 37 °C for 45 min. The digested tissues were then minced in a homogenized washing buffer containing 1× PBS with 1% FBS and collected. Hepatocytes were sedimented by centrifugation at 300×g. The remaining cells were separated into 4 layers by centrifugation at 500×g on a 35% Percoll (Sigma-Aldrich) gradient. Lymphocytes on the second layer were subsequently centrifuged at 250×g, resuspended in RBC lysis buffer and washed by complete RPMI 1640 medium. Finally, cells were passed through Pre-Separation Filters (20 μm; Miltenyi Biotec, Bergisch Gladbach, Germany) and were collected for further study.
Hepatic lymphocytes were prepared as described previously with some modifications [18 (link)]. The remaining hepatic tissues were washed in D-Hank’s solution (Invitrogen, Carlsbad, CA, USA) via the portal vein, cut into pieces and digested in 20 mg Collagenase B (Invitrogen) at 37 °C for 45 min. The digested tissues were then minced in a homogenized washing buffer containing 1× PBS with 1% FBS and collected. Hepatocytes were sedimented by centrifugation at 300×g. The remaining cells were separated into 4 layers by centrifugation at 500×g on a 35% Percoll (Sigma-Aldrich) gradient. Lymphocytes on the second layer were subsequently centrifuged at 250×g, resuspended in RBC lysis buffer and washed by complete RPMI 1640 medium. Finally, cells were passed through Pre-Separation Filters (20 μm; Miltenyi Biotec, Bergisch Gladbach, Germany) and were collected for further study.
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