Ketamine xylazine mixture

Ovariectomies were performed in prepubescent (P21) and adult Wistar rats (P42). For the surgery, animals were anesthetized by injection of an initial dose of 100 mg/kg ketamine and 7.5 mg/kg xylazine. Respiration, blink, and pinch reflex were observed throughout the surgery and, if needed, animals were injected with an extra shot (25%) of ketamine/xylazine mixture (Sigma-Aldrich, St. Louis, MO) or a 25% dose of ketamine (Sigma-Aldrich) alone. Monitoring of temperature was done using a rectal probe and could be maintained with a heating pad (Stoelting, Wood Dale, IL) to 34°C–36°C.
To remove ovaries a small incision was made bilaterally on the animal’s bag. The white fat pad, to which the ovaries are attached to, was pulled out through the body wall. Ovaries were grabbed with forceps and removed from the fallopian tube using small scissors. After successful removal, the white fat pad with fallopian tube was put back through the body wall. The incision was sutured using a self-resorbing thread. The wound was disinfected and looked after in the days following surgery.

Primary mouse hepatocytes were isolated as previously described56 (link)—in brief, mice were anaesthetized with a ketamine/xylazine mixture (Sigma), abdominal cavity exposed and inferior vena cava catheterized with a 23-gauge catheter (Becton Dickinson). The superior vena cava was clamped, portal vein was transected and Hepes-based perfusion solution infused into the liver for 2 min at 5 cc min⁻¹ followed by type I collagenase (Worthington Biochemicals), including protease inhibitor caplets (Boehringer Mannheim), for 18 min at the same flow rate. Liver cells were filtered, mixed with Percoll and spun at 100 g to remove dead cells and non-hepatocytes. After two washes, cells were plated at 0.8 × 10⁶ cells per well of six-well dish in Williams E supplemented with 5% FCS (Invitrogen). For inhibitor experiments, we treated hepatocytes with 20 nM rapamycin (Cell Signaling), 250 nM Torin1 or vehicle for either 1 or 24 h. The construct expressing HA/Ub was transfected using Lipofectamine 3000 (Invitrogen) according to the manufacture's recommendation.