In vivo experiment with poultry chicken

Two sets of chicken were kept for 36 days until the dissection took place and confocal microscopic images were obtained. From day 5, one set (n = 5) of poultry chicken was kept as control and the other set (n = 5) of chickens were treated (dosage 400 mg for first 15 days and 600 mg/day before dissection, dosage mentioned here administered orally with food, it is not the actual amount of intake)24 with colistin in food. Four times a day they were supplemented with commercially available food along with colistin (Purchased from local market as colistin sulfate). At the end of the treatment period (32 days) the chickens were sacrificed with ketamine anesthesia. Liver was perfused with the help of calcium-free perfusion buffer having EDTA and Hank’s solution. The liver was then dissected out, placed on a petri dish and perfused with the help of HEPES buffer containing 0.08% collagenase for 15 min. Liver pieces were then passed through sterile nylon mesh with complete Leibovitz-15 media. The hepatocytes were isolated by centrifugation at 50g × 2 min, washed 3 times with the Leibovitz-15 media and kept in incubator at 37 °C for 30 min; meanwhile we checked the cell viability.