Ultrasensitive insulin elisa kit

After C57BL/6 mice were fasted for 6 h, tail vein blood was collected. The samples were centrifuged at 3500 rpm for 10 min at 4°C to separate plasma. Plasma insulin levels were measured by enzyme-linked immunosorbent assay (ELISA) using an insulin ultrasensitive ELISA kit (ALPCO, Salem, NH, USA), according to the manufacturer's instruction. The concentrations of plasma triglyceride (TG) and total cholesterol (TC) were determined according to the kit instruction (Jian Cheng Biotechnology Company, Nanjing, China). Plasma glucose levels were detected to calculate the homeostasis model assessment (HOMA). HOMA-IR index = fasting blood glucose (mmol/L) × fasting plasma insulin (pmol/L)/22.5 [23 (link)]. The concentration of LDLR protein in liver tissues was assessed by ELISA kit according to the manufacturer's protocol. OD values were determined by enzyme microplate reader (Thermo Multiskan Mk3, Thermo Fisher Scientific Inc., Barrington, USA), with detection wave length of 450 nm. The LDLR level was calculated based on the standard curves.

Insulin secretion of islets and INS‐1 832/13 cells was determined as described previously.
²³ (link) Briefly, the cells were pre‐incubated in 1 mL KRB buffer for 1 hour, followed by stimulation with 16.8 mmol/L glucose or 6 mmol/L glucose in the absence or presence of 10 nmol/L GLP‐1 for 30 minutes. The insulin level was determined using the Insulin Ultrasensitive ELISA kit (ALPCO Diagnostics, Salem, NH).

Insulin and C-peptide secretion was measured in the culture medium of DIPCs and DIPC spheroids by static incubation for 2 days in differentiation culture medium containing 5.5 mM glucose. Additionally, DIPCs and spheroids were washed and lysed using RIPA lysis buffer to detect intracellular insulin contents [24 (link)]. Prior to glucose stimulation of cells, residual insulin released from cells was removed by incubating in serum- and glucose-free Krebs buffer. The tubes were incubated at 37 °C for 1 h with shaking; then, the medium was completely removed by centrifugation and replaced with Krebs buffer supplemented with 3 mM glucose for 1 h. After collecting the medium, the cells were incubated with Krebs buffer containing 30 mM glucose for 1 h. Supernatants were collected. The stimulation index was calculated by dividing the insulin concentration of the supernatant in response to the 30 mM glucose incubation by that of the supernatant from the 3 mM glucose incubation [25 (link)]. Insulin and C-peptide levels were measured using a commercial ultrasensitive insulin ELISA kit (ALPCO, Salem, NH, USA) and ultrasensitive C-peptide ELISA kit (Mercodia, Uppsala, Sweden), respectively.

Mice were fasted for 6 hours before performing an oral glucose tolerance test (OGTT) or an insulin tolerance test (ITT). For the OGTT, a glucose bolus of 2 g/kg body weight of 20% glucose solution was given by gavage. For the ITT, an insulin dose of 0.3 U/kg body weight was injected intraperitoneally. Glucose levels were measured with a One Touch Ultra glucose meter before the test and at 15, 30, 60, 90, and 120 minutes after gavage or injection. In addition, fasted insulin levels were measured with an ultrasensitive insulin ELISA kit (Alpco Diagnostics, Salem, NH). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated from fasted insulin and glucose levels (fasted insulin (μU/mL) × fasted glucose (mmol/liter)/22.5).

The phenotypes of homozygous KO (Senp2^fl/fl, RIP-Cre) male mice and their male littermates (Senp2^fl/fl) were compared. Mice were fed a normal chow diet (CD) or a high-fat/high sucrose diet (HFD) (58 Kcal % fat with sucrose, Surwit Diet D12331) for 12–15 weeks from the age of 7–8 weeks. An intraperitoneal glucose tolerance test (IPGTT) was performed at the age of 20 weeks in the CD-fed mice and after 12 weeks of HFD feeding. Mice were fasted overnight for 16 h, and D-glucose (2 g/kg body weight for mice on a CD and 1 g/kg for mice on a HFD) was injected intraperitoneally. Blood glucose levels were measured from the tail vein using a glucometer (Accu-Chek, Roche, Basel, Switzerland). For in vivo glucose-stimulated insulin secretion (GSIS), insulin levels were measured from the serum collected at different time points during the IPGTT by an ultrasensitive insulin ELISA kit (ALPCO, Salem, OR, USA). Proinsulin and C peptide levels were measured by ELISA kits (ALPCO).

Both ECs and ECCs were preincubated in Krebs-Ringer bicarbonate with HEPES buffer (KRBH; 115 mM NaCl, 24 mM NaHCO_3, 5 mM KCl, 2.5 mM CaCl_2, 1 mM MgCl₂, 25 mM HEPES) supplemented with 2% BSA (Sigma) containing 2.5 mM glucose (Invitrogen) at 37 °C for 2 h for equilibration. After an additional incubation in fresh buffer for 1 h, cells were incubated in KRBH containing 27.5 mM glucose (Invitrogen) or 2.5 mM glucose (Invitrogen) with 30 mM KCl (Sigma), 100 μM tolbutamide (Sigma) at 37 °C for 1 h. Secreted insulin was measured from the supernatant. To measure total insulin, the cells were lysed in 0.5 ml of acid-ethanol by sonication on a Vibra-Cell sonicator™ (Sonics & Materials, Inc., Newtown, CT, USA) for 40 sec; the cells were then neutralized with the same volume of 1 M Tris-HCl (pH 7.5). Secreted insulin and total insulin were measured using an Ultrasensitive Insulin ELISA Kit (Alpco, Salem, USA) according to the manufacturer’s instructions. A Mercodia C-peptide ELISA Kit (Mercodia, Sylveniusgatan 8A, Sweden) was used to measure secreted human c-peptide after the same procedure described above.

At the end of the study, blood was collected and centrifuged at 2000g for 10min for plasma separation. All samples were frozen at -80°C until analysis. Plasma insulin was measured with the Ultrasensitive Insulin ELISA Kit (ALPCO, Salem, NH, USA). Lipid profile was determined by quantifying plasma lipoproteins and lipids with commercial kits following the manufacturers’ instructions: triglycerides (Max Discovery, Austin, USA), LDL (Crystal Chem, Downers Grove, USA), HDL (Crystal Chem, Downers Grove, USA) and total cholesterol (Max Discovery, Austin, USA). LBL plasma level was measured with the LBP ELISA Kit for various species (Hycult Biotech, Netherland) following the manufacturer’s instructions.