Mouse ultrasensitive insulin elisa kit

Whole body lean mass and fat mass were measured in avertin-anesthetized mice using dual energy X-ray absorptiometry. Plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels were determined enzymatically according to the manufacturer’s guidance (Applygen). For the glucose tolerance test, mice were fasted overnight for 16 h and then injected intraperitoneally with 1.5 g/kg body weight glucose. For the insulin tolerance test, mice were fasted for 6 h starting at 8 am and then injected with 1 IU/kg body weight insulin. Blood glucose levels were measured with a portable glucometer (Accu-Chek, Roche) at 0, 15, 30, 60, and 120 min after injection. Serum insulin levels were measured by Mouse Ultrasensitive Insulin ELISA kits (Alpco) as described before [17 (link)].

After a 6 h fast, mice were injected with insulin (0.75 U kg⁻¹, i.p.; Sigma, St Louis, MO, USA). The blood glucose value and AUC were assessed before and at 30, 60, 90 and 120 min after injection of insulin. Blood serum was collected before glucose injection to measure the insulin concentration. insulin was measured using ALPCO mouse ultrasensitive insulin ELISA kits (Salem, NH, USA) according to the manufacturer's protocol.

Random blood glucose was determined in the morning between 7 and 10 am with a Contour glucometer (Bayer, Leverkusen, Germany). Plasma insulin levels were determined by Mouse Ultrasensitive Insulin ELISA kit (Alpco, Salem, United States).

Fasting blood glucose was monitored fortnightly in all mice as a health monitoring parameter using a hand-held glucometer (Accu-Chek Performa, Roche) from whole blood collected via saphenous tail vein bleeds. Mice were fasted for 3 h prior to blood collection. Two weeks before end-point, a glucose tolerance test was performed. Baseline fasting blood glucose levels were obtained and mice were then injected IP with 0.8 g/kg glucose solution. Further measurements were taken at 15, 30, 45, 60, 90 and 120 min post-injection via tail vein bleeds. In addition, glycated haemoglobin (HbA1c) was assessed at the experimental endpoint. Mice were anaesthetised with isofluorane prior to transcardiac puncture with a heparinised 26G needle (100 I.U/mL). A small aliquot of whole blood (20 μL) was used for HbA1c analysis, via the Cobas B 101 (Roche Diagnostics). Plasma was collected from the remaining blood and stored at − 80 °C until required. Insulin levels were evaluated from plasma using a mouse ultrasensitive insulin ELISA kit (Alpco, Beijing, China) in single replicates according to the manufacturer’s instructions.

For fasting glucose and insulin tests, blood was collected from the tail vein of mice after 5 h of fasting. The concentration of glucose was measured by a glucometer (Bayer Contour, Tarrytown, NY, USA). The concentration of insulin was determined by a Mouse Ultrasensitive insulin ELISA Kit (ALPCO Diagnostics, Salem, NH, USA). HOMA‐IR was calculated using the following formula: HOMA‐IR = fasting insulin (mU L⁻¹) × fasting blood glucose (mmol L⁻¹)/22.5.^[83] In addition, a glucose tolerance test was performed after overnight fasting. After injecting the mice with D‐glucose (1 g/kg body weight, i.p.), blood was collected from the tail vein at 0, 15, 30, 60, 90, 120 min post‐injection for the measurement of glucose levels.

Isolated mouse islets were incubated in Krebs-Ringer bicarbonate buffer (KRB, 111 mM NaCl, 4.8 mM KCl, 25 mM NaHCO₃, 2.3 mM CaCl₂, 1.2 mM MgSO₄, 0.15 mM Na₂HPO₄, 1.2 mM KH₂PO₄, 10 mM HEPES, and 0.2% BSA) containing 2.8 mM glucose for 2 hr with 0.05% DMSO, 5 μg/ml Nocodazole, or 5 μM Diazoxide. Islets were then transferred to fresh KRB with the corresponding chemicals plus 2.8 mM or 20 mM glucose with or without 40 mM KCl and incubated for 30 min. The supernatant was collected and the insulin content was determined using the Mouse Ultrasensitive Insulin ELISA Kit (ALPCO, Salem, NH, Cat#: 80-INSMSU-E01). One islet equivalent (IEQ) is defined as a spherical islet with a diameter of 150 µm and is equal to 1,767,146 µm³.