Mouse insulin elisa kit

Five to seven-week-old ICR male mice were purchased from Japan SLC. KLF15KO mouse was kindly gifted from Prof. Jain MK and genotyped as previously described (Fisch et al., 2007 (link)). All animals were maintained in a temperature-controlled environment with a 14-h-light / 10-h-dark cycle and were given free access to standard laboratory diet and water. For the fasting group, animals were starved 24-h, and for the refeeding group, they were re-fed for 16-h after a 24-h starvation. For the experiments of insulin-depleted diabetic mice, mice were administrated with streptozotocin (two intraperitoneal injections of 100 mg/kg body weight with 1-day interval) as previously described (Takeuchi et al., 2007 (link)). Plasma insulin levels were measured using mouse insulin ELISA KIT (Fujifilm). Liver triglyceride levels were measured using triglyceride E-test Wako kit (Fujifilm) as described previously (Yahagi et al., 1999 (link)). Mice were sacrificed in the early light phase in a fasted, re-fed state. All animals studied were anesthetized and euthanized according to the protocol approved by the Tsukuba University Animal Care and Use Committee. All experiments were repeated at least twice.

MIN6 cells were seeded into a 24-well plate at 5.0 × 10⁵ cells/well and maintained for 2–3 days. After overnight serum starvation, cells were pre-incubated for 2 h in Krebs–Ringer bicarbonate containing HEPES (KRBH) buffer (120 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.4 mM CaCl₂, 1.2 mM MgCl₂, 20 mM NaHCO₃, and 10 mM HEPES) containing 3 mM glucose and 0.5% (v/v) bovine serum albumin (BSA) at pH 7.4. Cells were incubated in 3 or 20 mM glucose-containing KRBH buffer for 1 h, and the culture supernatant was recovered. Insulin and EGF levels were measured using a mouse insulin ELISA kit (FUJIFILM Wako Shibayagi) and a mouse EGF ELISA kit (Thermo Fisher Scientific), respectively, according to the manufacturers’ instructions.

At the end of the experiment, the mice were fasted overnight and sacrificed by CO₂ asphyxiation. Blood was collected from the mice, stored at room temperature for 30 min, and centrifuged at 3000 rpm at 4 °C for 10 min to obtain serum. The concentrations of serum triglyceride (TG) and total cholesterol (TC) were assayed enzymatically using commercial kits (Asan pharms, Co., Seoul, Korea). Serum insulin content was detected using the mouse insulin ELISA kit (FUJIFILM, Co., Santa Clara, CA, USA). The insulin concentrations were expressed as mU/mL. Insulin resistance was measured with the homeostasis model assessment of insulin resistance (HOMA-IR), and was calculated using the formula: HOMA-IR = {Fasting glucose (mg/dl) × Fasting insulin (μU/L)}/405 [21 (link)].