Serum samples were analysed to determine the insulin levels using Ultra-Sensitive Insulin ELISA kit (Mercodia, Uppsala, Sweden).
Ultrasensitive insulin elisa kit
Manufactured by Mercodia
Sourced in Sweden
The Ultrasensitive Insulin ELISA kit is a laboratory assay designed to quantify insulin levels in biological samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) technique to provide a sensitive and accurate measurement of insulin concentrations.
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Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Male swiss mice, by Charles River Laboratories (1 mentions)
Colorimetric assay kit, by Abcam (1 mentions)
Triglyceride colorimetric assay kit, by Elabscience (1 mentions)
Nefa colorimetric assay kit, by Elabscience (1 mentions)
Ultra sensitive mouse insulin elisa, by Crystal Chem (1 mentions)
14 protocols using ultrasensitive insulin elisa kit
1
Insulin Levels Determination by ELISA
2
Streptozotocin-Induced Diabetic Mouse Model
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Male Swiss mice obtained from Charles River Laboratories (L’arbresle, France) were housed at constant temperature (20–22°C) and with a 12-h light–dark cycle. At the age of 2 mo, they received an intraperitoneal injection of STZ (40 mg/kg) diluted in citrate buffer (0.05 mmol/L, pH 4.5) for four consecutive days, as described previously[21 (link)]. A week later, mice receiving only citrate buffer (nondiabetic) and treated mice exhibiting blood glucose ≥ 300 mg/100 mL (STZ diabetic) were subdivided into four groups of eight males, with either free access to water (control) or a 0.5% Bza solution as drinking liquid (Bza-drinking) for 24 d. To measure plasma insulin levels at the beginning of treatment, blood samples were withdrawn from tail vein then centrifuged and analyzed using Ultrasensitive insulin-ELISA kit (Mercodia, Uppsala, Sweden). All the mice had free access to food and water and were treated in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments)[29 (link)]. During this period, nonfasting blood glucose levels were determined every 3 d at 12:00 h (equivalent in the used circadian rhythm to 4 h after lights turned on) using an Accu-Check glucometer (Roche Diagnostics) on a blood drop withdrawn from the tail vein. Mice were killed after overnight fasting at the end of treatment and organs were collected and weighed.
3
Metabolic biomarkers in brown adipose tissue
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Plasma levels of insulin, glucose, triglycerides, cholesterol, and non-esterified fatty acid (NEFA) were measured with the following commercial kits: ultrasensitive insulin ELISA kit (Mercodia, Uppsala, Sweden), glucose liquid (Química Analítica Aplicada SA, Tarragona, Spain), triglyceride colorimetric assay kit (Elabscience, Houston, TX, USA), cholesterol liquid kit (Química Analítica Aplicada SA, Spain), and NEFA colorimetric assay kit (Elabscience, USA), respectively. The HOMA-IR index was calculated as fasting plasma insulin (mU/L) × fasting plasma glucose (mmol/L)/22.5.
The citrate synthase activity was measured in the BAT using a colorimetric assay kit (BioVision, Milpitas, CA, USA). Hepatic glycogen was quantified according to the manufacturer’s instruction of a commercial kit (Sigma-Aldrich, USA). In addition, the lipid content was quantified in the liver after extraction with chloroform, as previously described [23 (link)]. Briefly, the tissues were homogenized in chloroform/methanol (2:1) solution. After 3 h of shaking, Milli-Q water was added, and the organic layer was separated by centrifugation (16,000× g, 20 min) and dried overnight. Triglyceride concentration was measured as described above.
The citrate synthase activity was measured in the BAT using a colorimetric assay kit (BioVision, Milpitas, CA, USA). Hepatic glycogen was quantified according to the manufacturer’s instruction of a commercial kit (Sigma-Aldrich, USA). In addition, the lipid content was quantified in the liver after extraction with chloroform, as previously described [23 (link)]. Briefly, the tissues were homogenized in chloroform/methanol (2:1) solution. After 3 h of shaking, Milli-Q water was added, and the organic layer was separated by centrifugation (16,000× g, 20 min) and dried overnight. Triglyceride concentration was measured as described above.
4
Insulin Secretion Modulation by CKs, Glu^h, Palm
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To study how CKs, Gluh, Palm, and Palm+Gluh affected INS1.1E7 cells’ insulin secretion in vitro, once the cells had been grown to a 90% confluence in 6-well plaques with the above described medium containing 5.5 mM D-glucose, the medium was changed to a 2.2 mM glucose for 24 h, to reduce basal insulin secretion before adding CKs, Gluh, Palm, and Palm+Gluh to the culture media for 48 h. Then, cells were washed three times with a 2.2 mM glucose HBSS/Krebs buffer (HBBS) and incubated with the same solution for 60 min, to establish basal insulin secretion in the supernatants. Thereafter, a high glucose concentration (22 mM) HBBS was added to the cultures for 90 min. Finally, the new supernatants were collected again to measure insulin and determine GSIS.
The insulin concentration was measured using an Ultrasensitive Insulin ELISA kit (Mercodia AB, Uppsala, Sweden). Briefly, the supernatants were diluted 1:10 in a glucose-free HBSS. Then, 25 µl of each sample in duplicates were incubated for 2 h with the kit’s conjugated enzyme. After washing six times, the samples were incubated 15 min in the dark with the kit’s TMB substrate solution. Finally, the reaction was stopped by adding the corresponding solution, and the absorbance was determined at 450 nm. All the incubations were performed at RT.
The insulin concentration was measured using an Ultrasensitive Insulin ELISA kit (Mercodia AB, Uppsala, Sweden). Briefly, the supernatants were diluted 1:10 in a glucose-free HBSS. Then, 25 µl of each sample in duplicates were incubated for 2 h with the kit’s conjugated enzyme. After washing six times, the samples were incubated 15 min in the dark with the kit’s TMB substrate solution. Finally, the reaction was stopped by adding the corresponding solution, and the absorbance was determined at 450 nm. All the incubations were performed at RT.
5
Lipid and Glucose Biomarker Assessment
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After overnight fasting, blood samples were collected from each subject and centrifuged in the absence or presence of EDTA to obtain serum and plasma. High-density lipoprotein cholesterol (HDL-C) was measured after precipitation of apoproteins B (apo B) containing lipoproteins27 (link); total cholesterol (Tot-C) and triglycerides levels were assayed in serum by the Trinder method. The coefficient of variation was <2% for Tot-C and HDL-C and <5% for triglycerides for intra-batch and inter-batch, respectively. Friedewald’s formula calculated low-density lipoprotein cholesterol (LDL-C) plasma levels. Plasma glucose (PG) was measured using standard enzymatic methods (FAR S.R.L., Italy). The coefficient of variation was <3% for intra-assay. Fasting insulin levels were assayed using an ultrasensitive insulin ELISA kit (Mercodia AB, Sweden). The coefficient of variation was <3% for inter-assay. Insulin resistance was estimated with homeostasis model assessment (HOMA-IR).
6
Glucose-Stimulated Insulin Secretion Assay
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The islet function was evaluated using the previously reported GSIS method [33 (link)]. Following drug treatments, the islets were collected to perform the GSIS assay. In brief, 150 islet equivalents (IEQs) were initially incubated in KRBH buffer (composed of 129 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 5 mM NaHCO3, 10 mM Hepes, and 0.1% BSA) supplemented with 2.8 mM glucose for 1 h. Subsequently, the islets were placed in a KRBH solution containing 2.8 mM glucose (low) and incubated at 37 °C for 1 h, and the supernatants were collected. Following this, the islets were washed thrice with KRBH and then incubated in KRBH solution spiked with 16.7 mM glucose (high) at 37 °C for 1 h, and the supernatants were collected again. The insulin content in the supernatant samples was measured using the Ultrasensitive Insulin ELISA kit (Mercodia, Uppsala, Sweden) following the manufacturer’s instructions. The stimulation index was calculated using the following equation: Stimulation index = Chigh glucose/Clow glucose.Chigh-glucose was the secreted insulin concentration of islets under high-glucose stimulation, and Clow-glucose was the secreted insulin concentration of islets under low-glucose stimulation.
7
Glucose-Stimulated Insulin Secretion Assay
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GSIS was performed to study the ability of differentiated cells to release insulin in response to glucose, as previously described (Truchan et al., 2015 (link)). Briefly, at the end of the differentiation, the clusters were washed with PBS and equilibrated for 1h in Krebs buffer (NaCl 129mM, KCl 4.7mM, CaCl2 2.5mM, MgSO4 1.2mM, Na2HPO4 1mM, KH2PO4 1.2mM, NaHCO3 5mM, HEPES 10mM, and BSA 0.1% in distilled water) with 1.7mM glucose. Then, cells were incubated with Krebs buffer with 2.8mM and 16.7mM glucose, 1h each. Next, clusters were dispersed with TryplE for 5 min and cells were counted using a haemocytometer. The insulin in the supernatants was quantified using Ultrasensitive Insulin ELISA kit (Mercodia).
8
Pancreatic Islet Isolation and Insulin Secretion
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Pancreata were digested with type V collagenase (C9263; 1,5 mg/ml) for 11 min at 37°C as described (Annicotte et al., 2009, Rabhi et al., 2016), and separated on a density gradient, islets were handpicked under a dissection microscope and cultured overnight in RPMI-1640 (Gibco, 61870-010) containing 1mM FBS (Gibco, 10270-106) and Penicillin/streptomycin. For insulin secretion tests, ~30 islets were exposed to 2.8 mM or 20 mM glucose in Krebs-Ringer-bicarbonate HEPES buffer containing 0.5% fatty-acid-free BSA. Secreted insulin was measured 1h later using the Ultrasensitive Insulin ELISA kit (Mercodia). Data are expressed relative to total insulin content.
9
Comprehensive Metabolic Phenotyping Protocol
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Metabolic phenotyping experiments were performed according to the EMPRESS protocols (http://empress.har.mrc.ac.uk ) as previously outlined10 (link), 63 (link). Briefly, intraperitoneal glucose (2 g of glucose per kg of body weight) and insulin (0.75 U of insulin per kg of body weight) tolerance tests were performed on 16-h-fasted animals for IPGTT and 5-h-fasted animals for ITT. Glycemia was measured before and at different time after glucose and insulin injections using the Bayer Contour. Circulating insulin levels were measured using the Ultrasensitive Insulin Elisa kit (Mercodia). Oxygen consumption, carbon dioxide production, RER, food intake, and physical activity were measured continuously using Comprehensive Laboratory Animal Monitoring System (CLAMS) consisting of open circuit calorimeter and motion detectors. Body composition was measured by noninvasive quantitative MRI (EchoMRI700). ALT/SGPT Liqui-UV Test (Rate), AST/SGOT Liqui-UV Test (Rate) and Triglycerides LiquiColor™ Test (Mono) Reagents were purchased from Stanbio and used for ALT, AST and triglyceride dosage respectively.
10
Ultrasensitive Insulin Quantification in CSF
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Insulin from CSF was measured by ELISA using Mercodia ultrasensitive insulin ELISA kit in triplicate (Catalog No. 10-1132-01). Briefly, 25 μL of samples and calibrators (0.15–20 mU/L) were mixed with 100 μL of enzyme conjugate in a precoated 96-well plate and incubated on a plate shaker (800 rpm) for 1 h at room temperature. The plate was then washed and added by substrate and stop solution. The optical density was read at 450 nm with a plate reader.
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