ion was filtered sequentially with 100 mesh, 200 mesh, and 500 mesh cell sieves. Then, the hepatocyte suspension was washed twice with basic medium. The cell density was adjusted to 16106 cells/mL with adherent culture medium. The hepatocyte suspension was seeded Materials and Methods Materials Fetal bovine serum, collagenase IV, HepatoZYME medium and RPMI-1640 medium were purchased from Gibco. Insulin and HEPES were obtained from SigmaAldrich. Dexamethasone acetate, vitamin C, ascorbic acid, penicillin, streptomycin, and other chemicals were obtained from Baoman Biotechnology. BML-275 was purchased from Santa Cruz Biotechnology, Inc.. Cell culture Hepatocytes were isolated using the collagenase perfusion method and were obtained as 27216982 previously described. The study protocol was approved by the Ethics Committee on the Use and Care of Animals, Jilin University. Briefly, the caudate lobe of the liver was obtained through surgical excision from a female Holstein calf anesthetized with thiamylal sodium under sterile conditions. The liver was perfused with perfusion solution to wash away the blood until the perfusion 
solution became clear. The liver was then perfused with a collagenase IV solution to digest the liver tissue. After digestion, the liver capsule was cut off. After a 4-h attachment period, the medium was replaced with growth medium containing 10% fetal bovine serum. The medium was replaced with fresh medium every 24 h. Hepatocytes were harvested with a cell Acacetin site scraper and transferred into a centrifuge tube. The cells were washed twice with ice-cold PBS and lysed with an SL-1000D ultrasonic cell disruption 8866946 apparatus. The lysate was centrifuged for 5 min at 120006g at 4uC, and the supernatant was used to determine the triglyceride content using an automatic biochemical analyzer. RNA extraction and real-time RT-PCR Hepatocyte RNA was extracted with a Takara RNA extraction kit according to the manufacturer’s instructions. RNA integrity was determined by electrophoresis on 1% agarose gels. The RNA was analyzed by spectrophotometry at 260 and 280 nm using a Gene Quant II RNA/DNA Calculator. Samples with an optical density ratio of RNA at 260/280 nm.1.8 were used for further analyses. The RNA was transcribed into cDNA using a reverse transcription kit according to the manufacturer’s protocol. Gene primers were designed using Primer Express software and shown in Acetic acid treatment Cells were serum-starved overnight and then treated with sodium acetate in the form of neutralized acetic acid to avoid changing the pH of the medium. The concentration of acetate was chosen according to the normal hematology standards of dairy cows. The hepatocytes were subjected to the following treatments. For time course experiments, hepatocytes were treated with 3.6 mM acetate for 0, 1, 3, 6, 10, 16, and 24 h. For doseresponse experiments, hepatocytes were treated with acetate and BML-275. The treated hepatocytes were divided into a control group, a low-dose acetate treatment group, a medium-dose acetate treatment group, a high-dose acetate treatment group, a BML-275 group, and a BML-275+acetate group; all groups were treated for 3 h. Each treatment concentration of acetate or BML-275 was replicated 24 times. Western blotting AMP and ATP level determination Hepatocytes were harvested with a cell scraper and collected directly into 0.5 mL ice-cold 6% perchloric acid. The cells were lysed by pipetting up and down repeatedly. The lysate was centrifuged for 5 m
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