Nicotine (CAS No. 54-11-5) and caffeine (CAS No. C0750) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol was obtained from Zhen Xin Co., Ltd. (Shanghai, China). The glucose colorimetric detection kit was purchased from Shanghai Rongsheng Biotech Co., Ltd. (Shanghai, China). The rat insulin enzyme linked immuno sorbent assay (ELISA) kit was provided by Mercodia (Uppsala, Sweden). The rat testosterone enzyme ELISA kit was provided by North Institute of Biological Technology (Beijing, China). The corticosterone (CORT) assay kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Total cholesterol (T-CHO), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) commercial kits were bought from Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) kits were purchased from Takara Biotechnology (Dalian, China). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). All other reagents were of analytical grade.
Rat insulin enzyme linked immunosorbent assay kit
Manufactured by Mercodia
Sourced in Sweden
The Rat Insulin Enzyme-Linked Immunosorbent Assay (ELISA) Kit is a laboratory product designed to quantitatively measure the concentration of insulin in rat biological samples. The kit utilizes the ELISA technique, which employs antibodies and color change to identify and quantify the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Acyl coa oxidase based colorimetric kit, by Roche (2 mentions)
Reverse transcription and real time quantitative polymerase chain reaction rt qpcr kits, by Takara Bio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rat tnf α elisa kit, by R&D Systems (1 mentions)
Irisin elisa, by BioVendor (1 mentions)
737 automatic analyzer, by Hitachi (1 mentions)
Glycerol, by Randox (1 mentions)
Cell culture insert, by Merck Group (1 mentions)
Bsa fraction 5, by Merck Group (1 mentions)
9 protocols using rat insulin enzyme linked immunosorbent assay kit
1
Biochemical Measurements in Rat Model
2
Metabolic and Inflammatory Markers in Rats
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Plasma glucose was determined using an enzymatic kit (Audit Diagnostics, Cork, Ireland). Plasma insulin concentrations were determined using a rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Mercodia AB, Uppsala, Sweden). Plasma leptin and adiponectin concentrations were measured using a rat leptin ELISA kit (Assay Designs, Inc., USA) and rat adiponectin ELISA kit (Chemicon International, Inc., California, USA), respectively. Plasma TNF-α concentration was determined using a rat TNF-α ELISA kit (R&D Systems, Inc., Minneapolis, USA). The plasma IL-6 and PAI-1 levels were determined using a rat IL-6 immunoassay ELISA kit (R&D Systems, Inc.) and Zymutest rat PAI-1 ELISA kit (Hyphen BioMed, Neuville Sur Oise, France), respectively. The homeostasis model assessment equation for insulin resistance (HOMA-IR) was expressed as an index of insulin resistance: fasting glucose concentration (mmol/L) × fasting insulin concentration (mU/L)/22.5. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using an enzymatic kit (Randox Ltd, Antrim, UK) and ALT enzymatic kit (Randox Ltd), respectively.
3
Glucose-stimulated insulin secretion
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A subset of islets (n = 10) from each experimental condition were washed extensively and incubated in Krebs Ringer bicarbonate (KRB) solution with 10% FBS and 4.4 mmol/L of glucose (Sigma). Islets were then stimulated with KRB solution containing 10% FBS and 22.6 mmol/L of glucose. Each incubation step was performed for 90 min at 37°C in a humidified 5% CO2 atmosphere. Supernatants were then collected and stored at −80°C. Insulin measurements were performed using a rat insulin enzyme linked immunosorbent assay (ELISA) kit (Mercodia, Uppsala, Sweden). Results are expressed as a stimulated index (SI) defined as the ratio of stimulated versus basal insulin secretion.
4
Evaluating Glucose, Lipid, and Insulin Metabolism
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Blood glucose levels were measured by glucose oxidase assay (GLU GOD, Erba-Lachema, Czech Republic) using tail vein blood drawn into 5% trichloracetic acid and promptly centrifuged. Serum TAG concentrations were quantified using standard enzymatic methods (TG L 250 S, Erba-Lachema, Czech Republic). Non-esterified fatty acid levels (NEFA) were determined using an acyl-CoA oxidase-based colorimetric kit (Roche Diagnostics GmbH, Germany). Serum insulin concentrations were evaluated using a rat insulin enzyme-linked immunosorbent assay kit (Mercodia, Sweden). Irisin serum concentrations were measured using an irisin enzyme-linked immunosorbent assay kit (Irisin ELISA, BioVendor, Czech Republic). The insulin sensitivity index (ISI) was calculated from fasting insulin (FI) and fasting glucose (FG) as described previously [37 (link)]. ISI = 1/(FG*FI), where FG is expressed as mg/dl and FI as mIU/l.
5
Metabolic Profiling in Plasma
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The following metabolites, metabolic hormones and adipokines were measured in plasma: uric acid, urea, creatinine, aspartate transaminase (AST), alanine transaminase (ALT), γ-glutamyl transpeptidase (GGt), leptin, interleukin-6, tumor necrosis factor α (TNF-α), glucose, insulin, cholesterol, high-density lipoprotein cholesterol and triglycerides.
The metabolites were analyzed using commercial kits according to the manufacturer's instructions in a Hitachi 737 Automatic Analyzer (Hitachi, Tokyo, Japan). The levels of leptin, IL-6 and TNF-α were measured using commercial rat enzyme-linked immunosorbent assay kits (Abcam, Cambridge, UK). The plasma levels of insulin were measured using a commercial rat insulin enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden).
The metabolites were analyzed using commercial kits according to the manufacturer's instructions in a Hitachi 737 Automatic Analyzer (Hitachi, Tokyo, Japan). The levels of leptin, IL-6 and TNF-α were measured using commercial rat enzyme-linked immunosorbent assay kits (Abcam, Cambridge, UK). The plasma levels of insulin were measured using a commercial rat insulin enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden).
6
Rat Insulin Level Measurement
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Twenty-four hours after the last treatment, blood was collected and serum was separated by centrifugation. Insulin levels were measured using rat insulin enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden) in serum.11 (link)
7
Plasma Biomarker Measurement Methods
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Plasma glucose levels were measured by glucose oxidase assay (GLU GOD, Erba-Lachema, Brno, Czech Republic) and TAG, cholesterol, HDL-cholesterol (Erba-Lachema, Brno, Czech Republic), and glycerol (Randox Laboratories Ltd., Crumlin, UK) concentrations were quantified using standard enzymatic methods. Non-esterified fatty acid levels (NEFA) were determined using an acyl-CoA oxidase-based colorimetric kit (Roche Diagnostics GmbH, Mannheim, Germany). Plasma insulin concentrations were measured using a rat insulin enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden). MCP-1 and leptin concentrations were measured by rat multiplex enzyme-linked immunosorbent assay kit (Milliplex: RADPKMAG-80K, Merck KGaA, Darmstadt, Germany).
8
Measuring Insulin Secretion in Rat Islets
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To determine the in vitro potency of isolated rat islets, the insulin secretory response to glucose was measured using a modified method previously described (47) . Briefly, 20-30 islets were randomly transferred to a cell culture insert (Millipore Ltd., Billerica, MA, USA) with Krebs buffer (115 mM NaCl, 5.0 mM KCl, 2.3 mM CaCl 2 , 1.0 mM MgCl, 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , pH 7.4), 25 mM HEPES, 0.1% BSA fraction V (Sigma-Aldrich) containing 2.8 mM glucose, and incubated at 37°C for 1 h as preincubation. Thereafter, they were suspended three times for 60 min at 37°C in Krebs buffer with the addition of various glucose concentrations (basal I: 2.8 mM, stimulation: 28 mM, basal II: 2.8 mM, respectively). The supernatants were collected and stored at -20°C. The insulin level was measured using a Rat Insulin Enzyme-Linked Immunosorbent Assay kit (Mercodia Inc., Winston Salem, NC, USA). The stimulation index (SI) was calculated as the insulin released into the stimulation medium divided by insulin released into the basal I medium.
9
Evaluating Insulin Resistance and Dyslipidemia in Rodents
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Blood was collected by retro-orbital puncture from the ether-anesthetized rats and subjected to centrifugation to obtain the serum. Blood glucose levels and insulin levels were evaluated to assess hyperglycemic condition. Serum insulin levels were measured using rat insulin enzyme-linked immunosorbent assay kit (Mercodia, Sweden). IR was assessed by the homeostasis model assessment (HOMA), a mathematical model describing the degree of IR from fasting plasma glucose and insulin. HOMA-estimated IR was calculated by multiplying fasting plasma insulin (mU/L) with fasting plasma glucose (mmol/L) divided by 22.5.
Total serum cholesterol, triglycerides (TG), LDL cholesterol, VLDL cholesterol, HDL cholesterol, and atherogenic index (AI) were assessed to evaluate the development of dyslipidemia in the model.
Atherogenic index was calculated by using the formula of Schulpis and Karikas[11 (link)] Percentage of protection offered by the study drugs was calculated based on Dhandapani.[12 (link)]
Total serum cholesterol, triglycerides (TG), LDL cholesterol, VLDL cholesterol, HDL cholesterol, and atherogenic index (AI) were assessed to evaluate the development of dyslipidemia in the model.
Atherogenic index was calculated by using the formula of Schulpis and Karikas[11 (link)] Percentage of protection offered by the study drugs was calculated based on Dhandapani.[12 (link)]
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