Normal rat kidney proximal tubular cells (NRK-52E) are maintained in six-well 100 or 150-mm, or 150-mm plates in a humidified incubator gassed with 5% CO₂ and 95% air at 37°C in DMEM containing 5% fetal calf serum (FCS). Cells are grown to 60% confluence and divided into four treatment groups: 1) untreated (Untx), 2) CS, 3) CS+Mitoquinone (MitoQ), and 4) CS+DecylTPP. Untreated cells remained at 37°C in DMEM containing 5% FCS (group 1). CS is initiated by washing cells with cold PBS twice and storing them in UW/Viaspan solution alone (4 h at 4°C) (group 2), CS+Mitoquinone (1 μM) (group 3), or CS+DecylTPP (1 μM) (group 4). In separate experiments, cells are exposed to CS plus RW by replacing UW solution alone or UW solution containing Mitoquinone or DecylTPP with DMEM containing 5% FCS overnight (18 h at 37°C)^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
Male CD1 mice (30-35?g) or C57BL/6J mice (20-25?g) are used. Seven intraperitoneal injections of a supramaximal dose (50?μg/kg) of Caerulein, a CCK-8 analogue, are given on an hourly basis to induce hyperstimulation acute pancreatitis (CER-AP). Control mice receive equal volumes of PBS injection. In the Mitoquinone treatment groups, Mitoquinone at 10?mg/kg (dose 1) or 25?mg/kg (dose 2) is given at the first and third injections of Caerulein. Similarly, dTPP at 9.6?mg/kg (dose 1) or 24?mg/kg (dose 2) is given for the dTPP treatment group. Mitoquinone and dTPP are at the same molar concentration at doses 1 and 2. Mice are sacrificed at 12?h after the first Caerulein injection to collect samples. Bile acid-induced AP is achieved by retrograde infusion of TLCS into the pancreatic duct (TLCS-AP). After induction of anesthesia, TLCS applied using a mini infusion pump at a speed of 5?μL/min for 10 minutes. Successful infusion of TLCS into pancreas is demonstrated by a diffuse light blue colour (methylene blue) appearing in the pancreatic head. Control mice receive sham surgery without TLCS infusion. In the treatment groups, Mitoquinone (10?mg/kg) or dTPP (9.6?mg/kg) is given at 1?h and 3?h after TLCS infusion. Mice are sacrificed at 24?h after the TLCS infusion or sham surgery.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Mitchell T, et al. The mitochondria-targeted antioxidant mitoquinone protects against cold storage injury of renaltubular cells and rat kidneys. J Pharmacol Exp Ther. 2011 Mar;336(3):682-92.  [Content Brief]

  [2]. Huang W, et al. Effects of the mitochondria-targeted antioxidant mitoquinone in murine acute pancreatitis. Mediators Inflamm. 2015;2015:901780.  [Content Brief]