Adoptive Transfer of Splenocytes

Splenocytes were prepared according to a previous study [20 (link)]. At D5, AcGP mice were euthanized by cervical dislocation, and the spleen was removed immediately. The spleen was placed in 3 mL of ice-cold RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 2% fetal bovine serum (FBS) and minced using the plunger of a 1 mL injection syringe. The suspension was filtered through a 70 μm mesh cell strainer (Corning, Glendale, AZ, USA) into a 50 mL tube and washed with 5 mL of ice-cold PBS containing 2% FBS. The single-cell suspension was centrifuged at 500× g for 5 min at 4 °C. After removal of the supernatant, the pellet was incubated in 3 mL of red blood cell lysis buffer (Abcam, San Diego, CA, USA) for 3 min at room temperature, and hemolysis was stopped by adding 5 mL of ice-cold PBS containing 2% FBS. Splenocytes were pelleted by centrifugation at 500× g for 5 min at 4 °C, washed with ice-cold PBS containing 2% FBS, and resuspended in 3 mL of the same buffer. The suspension was then filtered through a cell strainer. A 200 μL aliquot of the suspension containing 1 × 10⁷ cells was injected intravenously into the tail vein of each naïve recipient mouse.