2.4. Preparation of necrotic H22 cells
H22 cells were adjusted to 2 × 106 cells/ml in RPMI 1640 complete media (supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FBS). The cell suspension was incubated under 5% CO2 condition at 37 °C for 3 weeks, without changing media or subculturing. After that, the cells were disrupted by four cycles of freezing/thawing followed by sonication