Rat mouse insulin elisa kit

Circulating insulin levels were quantified in blood sera using the Rat/Mouse Insulin ELISA Kit EMD Millipore (Cat. # EZRMI-13K), following the manufacture instructions. IL-1β levels in culture supernatants or in plasma were quantified using the IL-1β ELISA MAX^TM Deluxe Sets Biolegend (Cat. # 432605) following the manufactures instructions.

Blood samples, collected at the end of the treatments, were centrifuged at 1700 g for 10 min at room temperature and serum was stored at −20°C until use. By using commercial reagents (Siemens Healthcare Diagnostics s.r.l., Milan, Italy) and an automated biochemistry analyzer (Dimension EXL, Siemens Healthcare Diagnostics s.r.l.) the levels of basal glucose, triglycerides, total cholesterol, low density lipoproteins cholesterol (LDL), high density lipoproteins cholesterol (HDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined. The concentration of insulin and leptin was measured using ELISA kits (Rat/Mouse Insulin ELISA Kit; Mouse Leptin ELISA Kit, EMD Millipore Corporation, Darmstadt, Germany) according to the manufacturers' instructions. Approximate insulin resistance (IR) was calculated using the homeostasis model assessment (HOMA)-IR using the following formula: [glucose (mmol/L) × insulin (μU/mL)]/22.5 [28 (link)].

At baseline and week 11, six mice per group were fasted for four hours, and maximum 200 µL EDTA-blood was drawn from the saphenous vein of the hind leg, centrifuged for 15 min at 3000× g, and plasma was stored at −80 °C prior to analysis. The fasting blood glucose level was measured using FreeStyle Lite (Abbot Diabetes Care Inc., Alameda, CA, USA). Mice were then injected with an intraperitoneal dose of 20% glucose in phosphate buffered saline corresponding to 2 g glucose/kg body weight. Blood glucose levels were measured at 15, 30, 60, and 120 min after the intraperitoneal injection. Insulin was measured in plasma using the rat/mouse insulin ELISA kit from Merck (Kenilworth, NJ, USA).

The plasma glucose, triglyceride, cholesterol and urine glucose levels were determined by the enzymatic colorimetric method using a commercial kit (ErbaLachemas.r.o., Brno, CZ). Plasma insulin concentration was evaluated by the Sandwich ELIZA method using a commercial kit (Rat/Mouse Insulin ELISA kit, Merck Millipore, MA, USA). Renal function was estimated by the determination of serum and urine creatinine, serum blood urea nitrogen (BUN) levels and estimated glomerular filtration rate (eGFR). Serum and urine creatinine and serum blood urea nitrogen (BUN) levels were measured using an automatic biochemical analyzer at the Clinical Laboratory, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand. eGFR was calculated using the following equation:- $$\mathrm{eGFR}=\left(\mathrm{urine}\mathrm{creatinine}\times \mathrm{urine}\mathrm{flow}\mathrm{rate}\right)/\mathrm{serum}\mathrm{creatinine}$$

Insulin levels were measured using Rat/Mouse Insulin ELISA kit (Merck). Triglycerides and FFAs were measured by Triglyceride Quantification Colorimetric/Fluorometric Kit (Biovision). pLenti-GFP-Zeo was purchased from Addgene. Mutants were generated by using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). AKT, pAKT, pAMPK, and pACC antibodies were obtained from Cell Signaling Technology.

INS-1E cells or human islets were treated with dantrolene and sitagliptin for 24 hours. In addition dantrolene and sitagliptin were kept in KRB buffer during GSIS. INS-1E cells were incubated in KRB buffer with 0 mM glucose for 40 minutes at 37 degrees. Cells were then incubated with KRB buffer and 2.8 mM glucose for 1 hour followed by KRB with 16.7 mM glucose for 1 hour at 37 degrees. Supernatant was collected for insulin quantification at the end of each incubation. Insulin levels were quantified using the Rat/Mouse insulin ELISA kit from EMD Millipore. Cells were lysed at the end of the experiment and protein was quantified using a NanoDrop system from Thermo Scientific. Data was presented as total insulin level normalized to total protein.
Human islets from 3 different donors were hand picked and 8–10 islets were transferred to an eppendorf tube. Islets were incubated in KRB buffer with 3.3 mM glucose for 40 minutes. Islets were then exposed to either 3.3 mM glucose or 16.7 mM glucose KRB buffer and after one hour buffer was collected for insulin analysis. Insulin levels were measured using the Stellux human insulin ELISA kit from ALPCO. DNA was extracted from islets using the Qiagen DNeasy Blood and Tissue Kit per protocol. DNA was quantified using a NanoDrop system from Thermo Scientific. Data was presented as total insulin level normalized to total DNA.