4.2. Hepatocyte Isolation

Before sampling, fish were starved for 24 h. Hepatocytes were isolated from eight yellow catfish according to the previously reported method [63] with slight modification. Firstly, blood was removed from yellow catfish by cutting off the branchial arch, and disinfected with 75% alcohol. After blood removal, the liver was carefully excised from the abdominal cavity, placed onto a plastic Petri dish, and rinsed twice with phosphate-buffered saline (PBS, pH 7.4, 4 °C) containing amphotericin-B (25 μg/mL), streptomycin (100 μg/mL), and penicillin (100 IU/mL). Then, the liver was aseptically minced into 1 mm³ pieces with scalpel and scissors, and the tissue was digested by 0.25% sterile trypsin at room temperature on a shaker for 30 min, and the digestion was terminated with M199 medium containing 10% FBS every 5 min. The cell suspension was collected. Then, the isolated hepatocytes were purified through nylon sieve with 200 μm mesh size. Hepatocytes were put into a 15 mL sterile centrifuge tube and added with RCLB (red blood cell lysis buffer) at a ratio of 1:4 to remove excess red blood cells (RBC). Hepatocytes were centrifuged at low speed (1000 rpm/min for 5 min) and washed twice with PBS to remove debris. Finally, the purified hepatocytes were resuspended in M199 medium containing 1 mmol/l-glutamine, 5% (v/v) FBS, penicillin (100 IU/mL), and streptomycin (100 μg/mL). Hepatocytes were counted using a hemocytometer based on the Trypan blue exclusion method. Hepatocytes with more than 95% cell viability were used for the subsequent experiments. The hepatocyte cell suspension (CS) at the cencentration of 1 × 10⁶ cells per mL was plated into 25 cm² flasks.