Preparation of single cell suspension of rat hepatocyte
The animal experiments were performed in the Oriental Institute of Science and Technology, Cell and Mol. Therap. Lab. animal facility and surgery chamber. The fresh and laboratory acclimatized animals were anesthetized and then euthanized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). Fifty grams of rat liver tissue processed to have single cell suspension. A nylon mesh was used to scrape the tissue in DBSS Buffer. The scarpped product was centrifuged at 120×g . The cellular pellet was washed with constituted L-15 media. A final suspension of the cells was made in 10 ml of constituted L-15 media containing 1500 mg/l D-glucose, 1% penicillin and streptomycin, 20% FBS, 1% Glutamine. 650 µl of media containing cell was added to each petri plate comprising of 9350 µl of constituted L-15 media containing the required drug.
Three in vitro groups based on duration of exposure were considered for this study. . Grp 1-3hr, Grp 2-5 hr and Grp 3-12 hrs exposure. Each petri dish was provided with 650 µl of cell suspension (from stock) and each group comprised of 4 plates containing a total of 10 ml solution such as Plate-1 (control): 650µl of cell suspension+ 9250 µl buffer, Plate-2: DAS-100 µM+ buffer (making up to 10 ml), Plate-3: Chal-100µM+buffer (making up to 10 ml), and Plate -4: DAS+Chal 100µM each + buffer (making up to 10 ml).