Rbc lysis solution

Briefly, splenocyte suspensions were isolated from mice (3/group) by pushing the spleens through a wire mesh one day prior to the challenge. RBCs were removed using the RBC lysis solution (Sigma), and splenocytes were supplemented with RPMI-1640 medium including 20% FBS and an antibiotic solution (10,000 IU/mL penicillin and 10,000 μg/mL streptomycin). Cells were plated in 96-well flat-bottom plates at 100 μL/well (5 × 10⁵ cells/well). Subsequently, 100 μL/well of medium containing HA protein (10 μg/mL), PBS (negative control) or Concanavalin A (ConA, 5 μg/mL; Sigma) (positive control) was added at 37°C and 5% CO₂ for 72 h. The proliferative activity was measured using a CCK8 kit (Beyotime, CHINA). The SI was calculated as the ratio of the average OD₄₅₀ value of the wells containing antigen-stimulated cells to the average OD₄₅₀ value of the wells containing only cells with the medium. All assays were performed in triplicate. Passive secretion of cytokines (interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ) from collected supernatants was detected using specific ELISA kits (R&D). Procedures were performed per the manufacturer's instructions. For each plate, the OD₄₅₀ was transformed to a concentration by applying a linear regression formula, which was calculated from the standards of each kit.

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.

The isolation of splenocytes was performed as described previously [26 (link)]. Briefly, mice were euthanized on day 14 post immunization, and spleens were pushed through a 70 μm cell strainer in complete RPMI1640 medium to prepare a single-cell suspension under aseptic conditions. The cells were centrifuged at 500× g for 5 min, resuspended in 3 mL RBC lysis solution (Sigma, Rahway, NJ, USA) and incubated for 5 min to lyse the red blood cells. After two cycles of washing in complete RPMI 1640 medium, the splenocytes were counted and kept on ice until required.