We used rats instead of mice to evaluate the effect of immepip on serum glucagon concentration because a glucagon ELISA kit required 100 μL of serum to measure glucagon correctly. 10-week-old Wister rats were fasted for 24 h prior to the experiment and were anesthetized with isoflurane. Immepip (30 mg/kg) dissolved in normal saline or just normal saline was administered intraperitoneally. Blood samples were collected from the caudal vein before injection as the baseline and at 15 min after injection. Samples were centrifuged for 30 min at 5000 rpm at 4 °C and then the supernatant plasma was collected. Plasma glucagon concentration was measured using a glucagon ELISA kit (Wako).
Glucagon elisa kit
Manufactured by Fujifilm
Sourced in Japan
The Glucagon ELISA Kit is a laboratory equipment product designed for the quantitative determination of glucagon in biological samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) technique to measure the concentration of glucagon.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Nacalai Tesque (2 mentions)
Ripa buffer, by Nacalai Tesque (2 mentions)
Lbis porcine insulin elisa kit, by Fujifilm (2 mentions)
Immepip, by Merck Group (1 mentions)
Histamine, by Merck Group (1 mentions)
Ultra sensitive mouse insulin elisa kit, by ALPCO (1 mentions)
Surestep blood glucose meter, by LifeScan (1 mentions)
D glucose, by Merck Group (1 mentions)
Stat strip xp2, by Nipro (1 mentions)
7 protocols using glucagon elisa kit
1
Immepip's Effect on Rat Glucagon
2
Porcine Islet Insulin and Glucagon Secretion
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Glucose-stimulated insulin secretion (GSIS) and glucagon secretion (GSGS) of porcine islets and islet organoids and islets was assessed by treatment with various concentrations of glucose. In brief, 300 islet equivalents (IEQs) and organoids were preincubated with 3.3 mM glucose for 60 min. After preincubation, the islets were stimulated with glucose at 3.3 mM (low glucose) or 16.5 mM (high glucose) for 60 min. Insulin and glucagon in culture supernatants were measured using an LBIS Porcine Insulin enzyme-linked immunosorbent assay (ELISA) Kit (Fujifilm Wako Shibayagi Co., Shibukawa, Japan) and a Glucagon ELISA Kit (Wako), respectively.
3
Quantifying Insulin and Glucagon in Organoids
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Internal insulin and glucagon were extracted from 300 IEQs and organoids using 1 mL RIPA buffer (Cat#16488-34; Nacalai Tesque, Kyoto, Japan) containing × 100 protease and phosphatase inhibitor cocktails (Cat#07575-51 and Cat#07574-61; Nacalai Tesque). The insulin and glucagon contents were measured using an LBIS Porcine Insulin ELISA Kit and a Glucagon ELISA Kit (Wako), respectively.
4
Glucagon secretion regulation by histamine
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αTC1.6 cells were seeded into a 24-well plate at a density of 2.0 × 105 cells per well and then preincubated in 500 μL of Krebs–Ringer bicarbonate buffer (KRB) containing 20 mM glucose for 30 min at 37 °C in 5% CO2. Subsequently, αTC1.6 cells were incubated in 500 μL of KRB containing 20 or 2.8 mM glucose ± 1 μM histamine (Sigma), 1 and 100 μM of the selective H3R agonist immepip (Sigma) [24,25] , or the selective H3R inverse agonist JNJ-5207852 (kindly gifted from Dr. Nicholas Carruthers, Johnson & Johnson Pharmaceutical Research and Development, USA) [26,27] , or KRB containing 20 mM KCl ± 1 μM immepip, for 1 h at 37 °C in 5% CO2. immepip and JNJ-5207852 were dissolved in distilled water. We used a glucagon ELISA kit (Wako) to measure glucagon concentration in KRB.
5
Glucose Homeostasis in Fasted Mice
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After fasting for 8 h, blood glucose concentrations were measured by a Surestep Blood Glucose meter (Lifescan, Milpitas, CA, USA), and any blood glucose concentrations higher than 500 mg/dL were not included. IVGTTs were performed by tail vein injection of D-glucose (1 g/kg; Sigma) on days 7 and 14. After fasting for 8 h, plasma insulin concentrations were determined by using an ultrasensitive mouse insulin ELISA kit (Alpco, Salem, NH, USA), following the manufacturer’s instructions. After fasting for 8 h, plasma glucagon levels were determined by using a glucagon ELISA kit (Wako Pure Chemical Industries, Osaka, Japan).
6
Insulin and Glucagon Quantification
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Insulin and glucagon were extracted from 300 IEQs using 1 mL RIPA buffer (Cat#16488-34; Nacalai Tesque, Kyoto, Japan) containing ×100 protease and phosphatase inhibitor cocktails (Cat#07575-51 and Cat#07574-61; Nacalai Tesque). The insulin and glucagon concentrations were measured using an LBIS Porcine Insulin ELISA Kit and a Glucagon ELISA Kit (Wako), respectively.
7
Plasma Metabolite Profiling in Mice
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Blood samples were collected after 6 h-fasting at the end of each experiment. Plasma glucose levels were determined using Nipro Statstrip XP2 (Nipro, Osaka, Osaka, Japan). Plasma lipid parameters were assessed using an enzymatic colorimetric assay (Fujifilm Wako Pure Chemical, Osaka, Osaka, Japan). Plasma levels of insulin and glucagon were measured by enzyme-linked immunosorbent assays (Ultra Sensitive “PLUS” Mouse Insulin ELISA Kit, Product ID M1105; Morinaga, Yokohama, Kanagawa, Japan; Glucagon ELISA Kit, Product ID 292-90001; Fujifilm Wako Pure Chemical).
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