After 12 h of food deprivation, rats were anesthetized using 10% chloral hydrate (0.03 mL/kg), abdominal aortic blood samples then centrifuged at 3500G for 10 min for plasma collection. Plasma samples were stored at −80 °C until further use. Fasting blood glucose (FBG) was determined by Glucose Oxidase method, and fasting blood insulin (FINS) level was determined by radioimmunoassay to evaluate insulin sensitivity at body level by Homoeostasis model assessment-insulin resistance index (HOMA-IR). The formula for calculation is as follows: HOMA-IR = FBG × FINS/22.5. Insulin ELISA kit (Cloud-Clone, China) was used to detect serum insulin levels.
Insulin elisa kit
Manufactured by Cloud-Clone
Sourced in China
The 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 detect and measure insulin concentrations. The kit provides the necessary reagents and protocols to perform this analysis.
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6 protocols using insulin elisa kit
1
Insulin Sensitivity Evaluation in Rats
2
Glucose and Insulin Tolerance Tests
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For the GTT, an oral gavage of 50% d -glucose (3 mg/g body wt; Sigma) was performed after 16 h fasting. Blood glucose was measured at 0, 15, 30, 45, 60, and 120 min via the medial metatarsal vein. For the ITT, human insulin (1.5 mU/g body wt; Novonordisk) was intraperitoneally injected after the birds underwent 3 h of fasting. Blood glucose was measured at 0, 15, 30, 45, and 60 min via the medial metatarsal vein. Glucose and insulin were measured using an Accu-Check blood glucose meter (Roche Diagnostics) and an insulin ELISA kit (Cloud-Clone), respectively.
3
Fasting Plasma Insulin and Glycogen
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Fasting plasma was obtained from each species. Insulin concentrations were detected using insulin ELISA kit (Cloud-Clone), and muscle glycogen were determined with a relative assay kit (Solarbio) according to the manufacturer’s instructions.
4
Glucose and Insulin Tolerance Tests
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For the GTT, an oral gavage of 50% D-glucose (3 mg/g body wt; Sigma) was performed after 16 h fasting. Blood glucose was measured at 0, 15, 30, 45, 60, and 120 min via the medial metatarsal vein. For the ITT, human insulin (1.5 mU/g body wt; Novonordisk) was injected to 2 millimetres deep of the enterocoelia using microsyringe after the birds underwent three hours of fasting. Blood glucose was measured at 0, 15, 30, 45, and 60 min via the medial metatarsal vein. Glucose and insulin were measured using an Accu-Check blood glucose meter (Roche Diagnostics) and an insulin ELISA kit (Cloud-Clone), respectively.
5
Insulin Secretion in miR-17-5p Modulated INS-1 Cells
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The INS-1 cells were transfected with negative control or miR-17-5p mimic as described above, respectively, in normal (5 mmol/L) or high glucose (25 mmol/L) conditions for 72 h. Then, cells were preincubated with Krebs-Ringer bicarbonate HEPES Buffer (KRBH) (Gibco, USA) containing 2.8 mmol/L glucose for 2 h. For insulin stimulation, cells were transferred to a new KRBH buffer with 16.7 mmol/L of glucose and incubated for 1 h at 37°C. Insulin concentration was determined relying on an insulin Elisa kit (Cloud-Clone Corp, Wuhan, China) in the supernatant.
6
Avian Glucose and Insulin Quantification
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To quantify circulating glucose levels, fasting plasma was obtained from 8 birds from each species and was measured using an Accu‐Chek blood glucose meter (Roche Diagnostics). Insulin content of fasting plasma was performed using insulin ELISA kit (Cloud‐Clone), lactate in plasma and muscle glycogen were determined with a relative assay kit (Solarbio) according to the manufacturer's instructions. More information about enzyme activity is presented in Supporting Information.
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