To test if the hormonal release of IPC, GPC cells and aggregates was glucose-dependent, two glucose concentrations (2 mM and 25 mM) were assayed. After pre-incubation with Krebs–Ringer buffer (KRB) without glucose (120 mM NaCl, 5 mM KCl, 2,5 mM CaCl2, 1,1 mM MgCl2, 25 mM NaHCO3, 10 mM HEPES, 0.1% BSA) at 37 °C for 2 h, the cells were incubated with KRB containing 2 mM glucose at 37 °C for 4 h. To induce insulin release, the cells were incubated with KRB containing 25 mM glucose for another 4 h. Then, the respective conditioned media were collected and tested for the content of released insulin with human insulin (Mercodia, Uppsala, Sweden. Catalog number 10-1132-01) and human glucagon (Cusabio, Houston, TX, USA. Catalog number CSB-E09207h) ELISA kits.
Human insulin
Manufactured by Mercodia
Sourced in Sweden, United Kingdom
Human Insulin is a lab equipment product used to measure the concentration of insulin in human samples. It provides a quantitative analysis of insulin levels, which is an essential biomarker for monitoring various metabolic disorders, including diabetes.
Automatically generated - may contain errors
4 protocols using human insulin
1
Glucose-Dependent Hormonal Release in Pancreatic Cells
2
Quantifying Insulin and Glucagon Levels
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Human Insulin (Mercodia; Cat # 10-1113-01), human proinsulin (Mercodia; Cat # 10-1118-01), mouse insulin (Crystal Chem; Cat # 90080), mouse proinsulin (Mercodia; Cat # 10-1232-01), mouse glucagon (Crystal Chem; Cat # 81518), mouse LEGENDplex custom 10-plex (Biolegend) were measured using ELISA kits following manufacturer’s instructions.
3
Glucose and Insulin Clearance Assays
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All metabolic tests were performed after 6 h of fasting [32] . For insulin clearance during an oral glucose challenge, fasting blood glucose and blood samples (50 µL) were collected from the tail vein after 6 hours of fasting. Mice were then given a 4 g/kg glucose dose by oral gavage and subsequent blood samples (50 µL) were collected from the tail vein at 10, 60, and 120 minutes post-gavage. For insulin clearance during an insulin challenge, mice were given human insulin (1 U/kg, NovoRapid) or human C-peptide (50 µg/kg, Sigma) by intraperitoneal injection and blood samples were collected by tail vein sampling at 0, 5, 30, and 60 minutes post-injection. All blood samples were kept on ice after collection and then centrifuged at 10,000 g for 10 min at 4˚C. Plasma was collected into fresh tubes and stored at -80˚C. Mouse insulin and C-peptide were detected by multiplex ELISA (Millipore) kit in the plasma samples collected during the oral glucose challenge. human insulin (Mercodia) and human C-peptide (Millipore) were detected by ELISA kits in the plasma samples collected during the human insulin or human C-peptide challenges, respectively.
4
Insulin Sensitivity Assessment via OGTT
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Glucose (Human Glucose, Randox, UK) and insulin (Human Insulin, Mercodia UK) were assayed in plasma using commercially available kits according to the manufacturer's instructions and spectrophotometers, a Camspec M330B and a Biotek, Synergy HT multi-mode microplate reader respectively.
The AUC for both glucose and insulin concentrations during the OGTT were calculated using the trapezoid rule. Insulin sensitivity was estimated using the Cederholm index (Equation 1): Equation 1: ISICederholm = 75000 + (G0 -G120) x 180 x 0.19 x BW/120 X GMean x log (IMean)
Where BW = body weight, G0 and G120 = plasma glucose concentration at 0 and 120 minutes (mmol.l -1 ) respectively, and IMean and GMean = mean insulin (mU.l -1 ) and glucose (mmol.l -1 ) concentrations during the OGTT respectively.
The AUC for both glucose and insulin concentrations during the OGTT were calculated using the trapezoid rule. Insulin sensitivity was estimated using the Cederholm index (Equation 1): Equation 1: ISICederholm = 75000 + (G0 -G120) x 180 x 0.19 x BW/120 X GMean x log (IMean)
Where BW = body weight, G0 and G120 = plasma glucose concentration at 0 and 120 minutes (mmol.l -1 ) respectively, and IMean and GMean = mean insulin (mU.l -1 ) and glucose (mmol.l -1 ) concentrations during the OGTT respectively.
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