Five to 10 adult fish ventricles were dissected and cut into pieces with surgical scissors. Tissues were then transferred into a 1.5-ml tube containing 1 ml of HBSS buffer with collagenase/dispase (5 mg/ml; Roche) and collagenase 2 (5 mg/ml; Worthington) and put on a shaker at room temperature for 60 to 75 min. Samples were gently mixed by pipetting every 15 min. Ice-cold HBSS with 10% fetal bovine serum was used to wash the samples twice, and ACK buffer [0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA (pH 7.2)] was used to remove blood cells. All samples went through a 70-μm sterile cell strainer. Propidium iodide (1 μg/ml) was used to stain for dead cells. After PI staining, cells were acquired through an 11-color Attune NxT system (Life Technologies). Data were then analyzed by FlowJo software (Tree Star).

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