To assess glucose-stimulated insulin secretion (GSIS), β-TC6 cells were incubated in secretion buffer [NaCl 129, KCl 4.8, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO3 5.0, and HEPES 10 (all mmol/L) supplemented with 1 mg/mL bovine serum albumin, adjusted to pH 7.4] for an additional 60 min with 2.8 or 20 mmol/L glucose [5 (link)]. After collecting the supernatant for insulin measurement, the β-TC6 cells were lysed in intermediate RIPA Lysis Buffer (Beyotime) for later evaluation of the total protein content with BCA protein assay reagent kit (Cat. No. P0010, Beyotime) following the manual. Insulin levels in cell culture medium and β-TC6 cells were measured using a mouse/rat insulin ELISA kit (Linco Research) and standardized by every milligram protein per hour in each well.
Rat mouse insulin elisa kit
Manufactured by Linco Research
Sourced in United States
The Rat/Mouse Insulin ELISA Kit is a quantitative in vitro assay designed for the measurement of insulin levels in rat and mouse serum or plasma samples. The kit utilizes the sandwich enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the target analyte.
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Lab products found in correlation
One touch profile, by LifeScan (2 mentions)
Intermediate ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay reagent kit, by Beyotime (1 mentions)
C57bl 6j mice, by Charles River Laboratories (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Chloramphenicol, by Meilun (1 mentions)
Rat leptin elisa kit, by Linco Research (1 mentions)
Am251, by Cayman Chemical (1 mentions)
Exendin 4 ex4, by Bachem (1 mentions)
Cp55 940, by Cayman Chemical (1 mentions)
Arachidonyl 2 chloroethylamide, by Cayman Chemical (1 mentions)
Win55 212 2, by Cayman Chemical (1 mentions)
Gpo trinder kit, by Merck Group (1 mentions)
Free fatty acid quantitation kit, by Merck Group (1 mentions)
Rat bnp elisa kit, by Assaypro (1 mentions)
Mouse rat leptin elisa kit, by BioVendor (1 mentions)
Glucose assay kit, by BioCat (1 mentions)
18 protocols using rat mouse insulin elisa kit
1
Measuring Glucose-Stimulated Insulin Secretion
2
High-Stearic-Acid Diet and Metabolic Outcomes
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Twenty male C57BL/6J mice (6 weeks old) were purchased from the Beijing Vital River Laboratory Animal Technology Company (Beijing, China). After adaptive feeding for 1 week, these mice were randomly divided into control and high-stearic-acid diet groups (n = 10 per group), according to their body weights. The control diet (1025) and high-stearic-acid diet (HSD) (H10060) were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China) (Additional file 1 ). After 20 weeks of feeding the mice, islets and blood samples were collected for biochemical analysis. Fasting (12 h) serum glucose, total cholesterol, triacylglycerol, high-density-lipoprotein cholesterol, and low-density-lipoprotein cholesterol levels were calculated using an automatic analyzer (Hitachi-7100; Hitachi, Tokyo, Japan), kits for which were purchased from Biosino Biotechnology, Co. (Beijing, China). Serum insulin level was measured using a mouse/rat insulin ELISA kit (Linco Research, St. Charles, MO, USA).
3
Luciferase, PCR, and Insulin Assays
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Luciferase activity assay The luciferase activity was detected using a luciferase activity assay. See ESM Methods.
Quantitative PCR The mRNA and miRNA levels were determined by quantitative PCR (qPCR). See ESM Methods. The primer sequences are shown in ESM Table 2.
Western blot The western blot procedure has been described previously [25] . See ESM Methods.
Insulin measurements Insulin levels in mouse serum and cell culture media were determined by mouse/rat insulin ELISA kit (Linco Research, St Charles, MO, USA). To assess glucose-induced insulin secretion (GSIS), cells were incubated in secretion buffer (see ESM Methods) for an additional 60 min with 2.8 or 20 mmol/l glucose.
Statistics Values are expressed as mean ± SD. Multiple groups were analysed with one-way ANOVA followed by a Student-Newman-Keuls test. Two-group-only comparisons were carried out by t test. A two-sided p value < 0.05 was considered statistically significant. Repeated-measures ANOVA was used to analyse the glucose and insulin levels during IVGTT.
Quantitative PCR The mRNA and miRNA levels were determined by quantitative PCR (qPCR). See ESM Methods. The primer sequences are shown in ESM Table 2.
Western blot The western blot procedure has been described previously [25] . See ESM Methods.
Insulin measurements Insulin levels in mouse serum and cell culture media were determined by mouse/rat insulin ELISA kit (Linco Research, St Charles, MO, USA). To assess glucose-induced insulin secretion (GSIS), cells were incubated in secretion buffer (see ESM Methods) for an additional 60 min with 2.8 or 20 mmol/l glucose.
Statistics Values are expressed as mean ± SD. Multiple groups were analysed with one-way ANOVA followed by a Student-Newman-Keuls test. Two-group-only comparisons were carried out by t test. A two-sided p value < 0.05 was considered statistically significant. Repeated-measures ANOVA was used to analyse the glucose and insulin levels during IVGTT.
4
Plasma Biomarkers and Insulin Resistance
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Plasma glucose, triglyceride and total cholesterol concentrations were determined by the enzymatic colorimetric method using a commercial kit (Biotech, Bangkok, Thailand). The plasma insulin, leptin and adiponectin concentrations were determined using the Sandwich ELISA method (Rat/Mouse Insulin ELISA kit, LINCO Research, USA). The insulin resistance was assessed by the homeostasis model assessment of insulin resistance (HOMA-IR). The HOMA-IR was calculated as follows [26 (link)]:
5
Pancreatic Islet Isolation and Glucose-Stimulated Insulin Secretion
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Pancreatic islets were isolated by collagenase V digestion as previously described29 (link). For db/db mice, islets were cultivated overnight in DMEM supplemented with 10% FBS (fetal bovine serum), 10 hand-picked islets of similar size were incubated over a period of 1 h in 100 µl glucose-free Krebs-Ringer bicarbonate (KRB) buffer, and then treated for 1 h in KRB buffer containing 2.5 mM/L or 25 mM/L glucose. The supernatants were obtained for insulin concentration determination using a rat/mouse insulin ELISA kit (Linco Research, St Charles, MO). For Balb/c mice, handpicked islets were further cultivated in DMEM medium with or without 1 μg/ml doxycycline (Sigma-Aldrich, Shanghai, China), or 100 μg/ml chloramphenicol (Meilun Biological Technology, Dalian, China), or 1 μg/ml levofloxacin (P.D. Pharmaceutical, Guandong, China) before being used for GSIS measurement. Each experiment was independently repeated three times.
6
Rat/Mouse Insulin ELISA Assay
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The plasma insulin concentrations were measured by a Sandwich ELISA method (Rat/Mouse Insulin ELISA kit) as per the manufacturer’ instruction (LINCO Research Inc., St. Charles, MO, USA).
7
Glucose-Stimulated Insulin Secretion Assay
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After incubation, cells were washed with PBS. To measure glucose-stimulated insulin secretion (GSIS), we starved cells for 5 hours in DMEM containing 5 mM glucose and 2% FBS. Then the medium was replaced with KRBB solution (4.74 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgCl2·6H2O, 35 mM NaHCO3, 10 mM HEPES) containing 5 mM glucose or 25 mM glucose and the cells were incubated for 1 hour. To separate total insulin protein, we washed cells with PBS and lysed them with mammalian tissue lysis/extraction reagent containing a protease inhibitor. Insulin protein in the medium was measured using a Rat/Mouse Insulin ELISA Kit (Linco Research, St. Charles, MO, USA).
8
Rat/Mouse Insulin ELISA Protocol
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Serum insulin levels were measured using the following kits: a Rat/Mouse Insulin ELISA kit (Linco Research, St. Charles, MO, USA) in accordance with the manufacturers’ specifications.
9
Plasma Metabolite and Hormone Analysis
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EDTA plasma collected on P25, P45, and P95 rats were used to measure plasma glucose, NEFA (non-esterified fatty acids), insulin, and leptin. Glucose and NEFA were measured using colorimetric enzymatic reactions with specific kits (glucose and NEFA PAP 150 kits, BioMérieux, Marcy-l’Etoile, France). Hormones were assayed with specific ELISA kits following the manufacturer’s instructions for insulin and leptin (rat/mouse insulin ELISA kit, rat leptin ELISA kit, Linco Research, St. Charles, MO, USA).
10
Plasma Analysis of Metabolic Markers
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The blood samples (≈0.8 mL) were collected from the orbital plexus of rats under anaesthesia in heparinised microcentrifuge tubes. The plasma was separated by centrifugation and analyzed for glucose, triglycerides, total-cholesterol, and HDL-cholesterol using commercially available spectrophotometric kits (Accurex Biomedical Pvt. Ltd., India). Plasma insulin was estimated by rat/mouse insulin ELISA kit (rat/mouse insulin ELISA kit, Linco Research, USA) as per the manufacturer's instructions.
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