Gentlmacs dissociator

Spleens were pooled by pairs (5 pools group⁻¹) and were mechanically homogenized (GentlMACS Dissociator, Miltenyi Biotec, USA). After lysis of red blood cells with lysing buffer (Red Blood Cell Lysing Buffer Hybrid‐Max, Sigma‐Aldrich), splenocytes were resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM l‐glutamine, 100 U penicillin and 100 mg mL⁻¹ streptomycin. Cells (10⁶ cells well⁻¹) were incubated for 60 h at 37 °C (5% CO₂) in 96‐well culture plates in the presence of purified SFS protein (20 µg mL⁻¹) or PBS. Polymyxin B sulfate salt (50 µg mL⁻¹, Sigma‐Aldrich) was added to each activator for endotoxin neutralization. Cytokines secretion (IL‐4, IL‐5, IL‐10, IL‐13, and INF‐γ) was measured in duplicate in collected supernatants by using Bioplex 200 System and commercial multiplexed kits according to the manufacturer's instructions (Bio‐Rad, Marnes‐la‐Coquette, France).

Membrane proteins containing H-2K^b (K^b) were isolated from an EG7.OVA tumor mass. Briefly, 1 g of EG7.OVA tumor mass was dissociated with 10 ml of extraction buffer containing 0.15 M NaCl, 0.05 M sodium phosphate buffer (pH 7.0), and 1X protease inhibitor cocktail (Sigma-Aldrich) using gentlMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). After removing cell debris and lipids, the supernatant was ultra-centrifuged at 80,000 × g for 45 min at 4°C using Ultracentrifuge (Beckman Coulter, Brea, CA, USA). The pellet was homogenized in 0.5 ml of extraction buffer using Tissue Grinder (SHS-30E; SciLab, Seoul, Korea). The homogenate was then mixed with 0.5 ml of solubilization buffer containing 2% n-dodecyl β-D-maltoside and 0.4% 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate hydrate (Sigma-Aldrich) in extraction buffer and solubilized for 90 min at 4°C. The supernatant containing membrane proteins was obtained by centrifugation at 17,000 rpm for 30 min at 4°C. The amount of isolated membrane proteins was determined using a microbicinchoninic acid assay kit (ThermoFisher Scientific, Rockford, IL, USA).