The blood collection was performed on Mondays after providing ad libitum food during the weekends. Five hours prior to the blood collection, food was removed. Blood glucose measurements were directly performed with the glucometer Freestyle Lite (Abbott Diabetes Care, Hoofddorp, The Netherlands). Blood was centrifuged at 4000 rpm and 4 °C for 10 min, and plasma was collected and stored at −80 °C until further use. Plasma insulin levels were quantified using an Enzyme-Linked Immunosorbent Assay (ELISA) (Ultra-Sensitive Rat insulin ELISA, Crystal Chem Inc., Zaandam, The Netherlands), using 5 μL plasma in duplicate for each sample and executed according to the manufacturer’s instructions. The absorbance values were obtained with a 450 nm excitation filter and corrected with the absorbance value obtained from both a 630 nm excitation filter and the sample diluent. The corrected and averaged concentrations of 5 measurements (from week 4 to week 12) are presented as nanograms per milliliter.
Ultrasensitive rat insulin elisa
Manufactured by Crystal Chem
Sourced in United States
The Ultrasensitive rat insulin ELISA is a laboratory kit designed to quantify insulin levels in rat samples. It utilizes an enzyme-linked immunosorbent assay (ELISA) technique to detect and measure rat insulin concentrations with high sensitivity.
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Lab products found in correlation
Infinite m200 nanoquant reader, by Tecan (3 mentions)
Glomax discover microplate reader, by Promega (2 mentions)
Streptozotocin (stz), by Merck Group (2 mentions)
Freestyle lite, by Abbott (1 mentions)
Hdl cholesterol precipitating reagent, by Horiba (1 mentions)
Mouse leptin elisa kit, by Crystal Chem (1 mentions)
Onetouch ultra blood glucose meter, by LifeScan (1 mentions)
Tirzepatide, by Selleck Chemicals (1 mentions)
Forskolin, by Merck Group (1 mentions)
Exendin 4, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Bicinchoninic acid method, by Thermo Fisher Scientific (1 mentions)
Saccharin, by Merck Group (1 mentions)
Sucrose, by MP Biomedicals (1 mentions)
Dextrose, by Vedco (1 mentions)
9 protocols using ultrasensitive rat insulin elisa
1
Plasma Insulin Levels in Rats
2
Insulin Secretion in Pancreatic Islets
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Pancreatic islets of similar size were isolated from DNAJC3 K.O. or C57BL/6 wild-type littermates (control mice) and starved for 1 hour in Krebs Ringer HEPES (KRH) buffer. The buffer contained 15 mM HEPES, 5 mM KCl, 120 mM NaCl, 2 mM CaCl2, 10 μM glycine, 24 mM NaHCO3, and 1 mg mL−1 bovine serum albumin, supplemented with 2 mM glucose. To determine insulin secretion under basal and glucose-stimulated conditions, we measured the amount of insulin secreted from the same islets at 2 mM glucose (low glucose) and 20 mM glucose (high glucose). The islets were incubated in fresh low-glucose KRH buffer for 1 h, followed by incubation in high-glucose KRH buffer for 1 h. The supernatants were collected to determine the amount of insulin secreted. We then lysed the islets in RIPA buffer to measure insulin content. Insulin secretion and content were measured using an ultrasensitive rat insulin ELISA (Crystal Chem, Zaandam, The Netherlands) in combination with an Infinite M200 NanoQuant reader (Tecan, Männedorf, Switzerland). Insulin secretion was normalized to total insulin and expressed as a percentage of basal control insulin secretion. To measure the insulin content, the insulin was normalized to the total protein content, which was measured using the bicinchoninic acid (BCA) method (Thermo Fisher Scientific, Waltham, MA, USA).
3
Comprehensive Metabolic Profiling in Rodents
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Blood glucose was determined at baseline and weekly thereafter using the OneTouch Ultra blood glucose meter (LifeScan). Plasma insulin levels were analyzed using an ELISA for rat insulin (Ultra Sensitive Rat Insulin ELISA; Crystal Chem). Plasma leptin levels were determined using a mouse leptin ELISA kit (Crystal Chem). Quantitative determination of mouse MCP-1 levels in plasma was performed by using an ELISA kit (R&D Systems). Plasma cholesterol was determined using a cholesterol reagent colorimetric assay kit (Roche Diagnostics). For determination of plasma HDL cholesterol levels, plasma was incubated with an HDL cholesterol precipitating reagent (Pointe Scientific, Canton, MI) followed by separation of HDL by centrifugation (2,000g, 10 min). HDL was then quantified using an enzymatic cholesterol detection kit (Roche Diagnostics).
4
Glucose metabolism in DNAJC3 knockout mice
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To measure fasting blood glucose concentrations, we fasted 3-8-week-old DNAJC3 K.O. and C57BL/6 wild-type littermates (control mice) once a week for 16 h. We measured glucose concentrations twice in blood collected from the tail using the Monometer Futura glucometer (MedNet GmbH, Münster, Germany). For the glucose tolerance test, we injected 1.5 mg glucose/g body weight intraperitoneally (i.p.). Glucose concentrations were measured twice in blood collected from the tail before and at 15, 30, 60, and 120 min after i.p. injection. Insulin concentrations were measured before and 15 minutes after i.p. glucose injection using an ultra-sensitive rat insulin ELISA (Crystal Chem, Zaandam, The Netherlands) in combination with the GloMax Discover Microplate Reader (Promega, Madison, WI, USA).
5
Streptozotocin-Induced Diabetic Mouse Model
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Streptozotocin (STZ, Sigma-Aldrich) was dissolved in 10 mM citrate buffer, pH 4.5, and 40 mg STZ/kg body weight were immediately injected intraperitoneally (i.p.) into mice for five consecutive days [20 (link)]. Glucose tolerance tests were carried out by i.p. injections of 1 g glucose/kg body weight into mice after overnight starvation. Plasma insulin concentrations were measured in 14–16 weeks-old male DJ-1 KO and wild type mice after STZ treatment using an ultrasensitive rat insulin ELISA (Crystal Chem, Chicago, IL, USA).
6
Glucose-stimulated Insulin Secretion Assay
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The method has been previously described [12 (link)]. In brief, pancreatic islets were first starved in Krebs Ringer HEPES (KRH) buffer supplemented with 2 mM glucose ± Kom56. Subsequently, pancreatic islets were incubated in fresh KRH buffer ± Kom56 containing 2 mM glucose (low glucose) to measure the basal insulin secretion, and afterwards, the same pancreatic islets were incubated in fresh KRH buffer ± Kom56 containing 20 mM glucose (high glucose) to measure the glucose-stimulated insulin secretion (GSIS). For the insulin measurement after treatment with different glucose concentrations (2 mM, 5 mM, 10 mM, and 15 mM), the pancreatic islets were first starved in KRH buffer supplemented with 2 mM glucose ± Kom56. To determine the insulin secretion, the same pancreatic islets were subsequently incubated with fresh KRH buffer ± Kom56 and containing the respective glucose concentration. Kom56 and exendin-4 (Sigma–Aldrich) were dissolved in water, forskolin (Sigma–Aldrich) and tirzepatide (Selleckchem) were dissolved in DMSO (Sigma–Aldrich). Insulin concentrations were measured using an ultra-sensitive rat insulin ELISA (Crystal Chem) in combination with a GloMax Discover Microplate Reader (Promega).
7
Insulin Secretion Measurement in Pancreatic Islets
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To determine insulin secretion, pancreatic islets were starved for 1 h in Krebs Ringer HEPES (KRH) buffer (15 mM HEPES, 5 mM KCl, 120 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 24 mM NaHCO3 and 1 mg ml−1 bovine serum albumin) supplemented with either 2 or 2.5 mM glucose. The same islets were first incubated with fresh KRH buffer supplemented with 2 or 2.5 mM glucose (low glucose) for 1 h, followed by a 1 h incubation with KRH buffer supplemented with 5, 10 or 20 mM glucose (high glucose). Subsequently, the islets were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 0.25% Na-deoxycholate, 1% IGEPAL) to measure total insulin concentrations. Secreted and total insulin were measured using an ultrasensitive rat insulin ELISA (Crystal Chem) in combination with an Infinite M200 NanoQuant reader (Tecan). Secreted insulin was normalized to total insulin levels and presented as a percentage of insulin secretion at either 2.5 or 5 mM glucose.
For measurement of islet insulin content, total islet insulin content was normalized to total protein content which was measured by the bicinchoninic acid method (Thermo Scientific).
For measurement of islet insulin content, total islet insulin content was normalized to total protein content which was measured by the bicinchoninic acid method (Thermo Scientific).
8
Glucose Tolerance in Male Mice
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Glucose tolerance tests were performed on male mice after overnight fasting (16 h). Glucose was intraperitoneally (i.p.) injected at a concentration of 1 mg g−1 body weight, and blood glucose concentrations were measured before and 15, 30, 60, 90 and 120 min after glucose administration. Measurements were taken twice at each point of time using a Monometer Futura Glucometer (MedNet GmbH). Plasma insulin concentrations were measured before and 30 or 120 min after glucose injections using an ultra-sensitive rat insulin ELISA (Crystal Chem) in combination with an Infinite M200 NanoQuant reader (Tecan). No method of randomization was used. However, the animals of each group were chosen and placed in random order by the animal house staff. Investigators were not blinded.
9
Sucrose Consumption and Glucose Tolerance
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