Blood cell lysis buffer

First, 1 mL of sterile 4% thioglycolate broth was administered to male C57BL/6 mice via intraperitoneal (IP) injection. Peritoneal macrophages were collected through IP lavage four days after the injection. After an additional washing process with Blood Cell Lysis Buffer (Sigma), the isolated peritoneal macrophages were seeded in a 96-well plate with a concentration of 1 × 10⁶ cells/mL in 10% FBS and 1% antibiotics complemented RPMI 1640 at 37 °C in 5% CO₂.

Spleens were harvested in ice-cold RPMI-1640 medium (Gibco, Stockholm, Sweden) and stored on ice until processing within hours. Spleens were pressed against a 70 um nylon cell strainer (Falcon, New York, NY, USA) to obtain a single cell suspension of splenocytes, followed by a washing step with RPMI. The pellets were resuspended and mixed in blood cell lysis buffer (Sigma-Aldrich, St Louis, MO, USA) to remove erythrocytes. Cells were counted and suspended in complete RPMI containing 1 mmol/L sodium pyruvate, 10 mmol/L Hepes, 50 U penicillin, 50 µg/mL streptomycin, 0.05 mmol/L 2-mercaptoethanol, 2 mmol/L L-glutamine and 10% heat inactivated fetal bovine serum (FBS) all purchased from Gibco (Stockholm, Sweden). One million splenocytes were cultured in microtiter 48-well plates and stimulated for 18 h with anti-CD3/CD28 beads. Cells were spun down at 1000 g for 5 min at 4 °C and supernatants were stored at −80 °C until further analysis.