Isolation of neonatal rat ventricular cardiomyocytes (NRVMs)

NRVMs were isolated from postnatal day 1 hearts according to a previously described method²². Briefly, the ventricles of 1-day-old Sprague-Dawley rat pups were dissected and washed with HBSS without Ca²⁺ and Mg²⁺ (MacGene, CC016). Using micro-dissecting scissors, ventricular tissue was minced to obtain ~1 mm³ pieces, which were then treated with 5 mL of digestion solution containing collagenase II (0.3 mg/mL; Thermo, 17101015) and trypsin (1 mg/mL; Amresco, VWRV0785) in HBSS for 5 min at 37 °C. The resulting supernatants were removed and the residual tissue was repeatedly treated with the digestion solution until little remained. The cells in the supernatants were transferred to a tube with an equal volume of ice-cold DMEM containing 10% FBS and 1% penicillin–streptomycin and centrifuged at 1000 × g for 4 min at room temperature. The cell pellets were re-suspended in 25 mL DMEM containing 5% horse serum (MacGene, CS008), 1% penicillin–streptomycin, and 1 μmol/L cytosine arabinoside, and then incubated in a 100-mm dish for 1.5 h at 37 °C to eliminate fibroblast contamination. Non-adherent cells were collected and seeded at a final concentration of 5 × 10⁵ cells/mL. After incubation for 48 h, the medium was removed and the NRVMs were then cultured with DMEM containing 10% FBS and 1% penicillin-streptomycin. To co-culture NRVMs and neutrophils, equal numbers of ABNs and NRVMs were mixed and then incubated at 3 °C for 12 h before performing cell viability assays.