Rat insulin elisa kit

At PND60, blood samples (300 µL) were collected from 6 h fasted offspring. An oral glucose tolerance test (OGTT) was performed in offspring at PND61 and PND62. Following 6 hours of fasting, all rats received a 2 g/kg BW dose of glucose by gavage. Blood samples (100 µL) were collected to determine plasma insulin concentration before (T0) and after 15 (T15) and 30 min (T30) gavage with glucose. Blood glucose was measured at T0, T15, T30, T45, T60, T90, and T120 after glucose intake, using a Performa Accu-Chek® glucometer (Roche Diabetes Care France, Meylan, France).
Insulin (Rat Insulin ELISA kit®, ALPCO, Salem, USA), glucose (Glucose GOD FS®, DiaSys, Holzheim, Germany), triglycerides (Triglycerides FS®, DiaSys, Holzheim, Germany), and cholesterol (Cholesterol FS®, DiaSys, Holzheim, Germany) were measured in plasma, following manufacturers’ instructions. Optical density was read with a microplate reader Varioskan Lux® (ThermoFisher Scientific, Waltham, USA).

RINm5F cells (200,000/well) were seeded in 24-well plates with a supplemented RPMI medium and allowed to confluence. The medium was then changed by Ringer-Krebs buffer (115 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 24 mM NaHCO₃, 10 mM HEPES, 1 g/L BSA, and 1.1 mM glucose, pH 7.4) for 2 h to leave the cells in a fasting state. Subsequently, the medium was replaced with another medium that was free of serum, containing the fractions of the aqueous alkaloid-free extract from C. roseus (aqueous fraction and precipitate; 10 and 100 μg/mL, respectively) and glibenclamide (4 μM). The medium was collected after 60 min and the insulin concentration was measured with the ALPCO™ Rat Insulin ELISA kit following the supplier's instructions.

Insulin and estradiol serum concentrations were determined using enzyme-linked immunosorbent assay (ELISA) kits (ELISA; Rat Insulin ELISA Kit, ALPCO; 17-β Estradiol ELISA Kit, Abcam). Blanks, standards, and serum samples were added to a 96-well microplate precoated with anti-insulin or anti-estradiol IgG. Insulin or 17-β estradiol-HRP conjugate was added to each well, and plates were incubated for 2 hours at 25-37°C. Following incubation, plates were aspirated and washed three to six times with wash buffer. The substrate solution was then added to each well and further incubated for 15-30 minutes. Following substrate incubation, stop solution was added to each well, and plates were immediately read at 450 nM using a microplate reader. The absorbance values of samples were adjusted based on the absorbance of blanks. Hormone concentration was interpolated from a standard curve generated from the blank and standards.

Blood extraction (1 mL) for biochemical measurements was performed after 12 h of fasting via cardiac puncture under anesthesia (0.3 mg/kg body weight). Both biochemical and physiological parameters in all the study groups were performed at day 0 (under basal normoxic conditions) and after 30 days (immediately after descending from the chamber). The hematocrit (Hct) and hemoglobin (Hb) values were measured. Serum insulin was measured using a commercial kit (Rat Insulin ELISA Kit^®, ALPCO, Salem, VT, United States), and glucose was measured using a glucometer (CarenSensN^®). The HOMA2 model was used to calculate the sensitivity (HOMA2%S) index with the HOMA2 calculator version 2.2 (Diabetes Trial Unit, University of Oxford), and body weight and residual food were measured every 4 days using an electronic scale (Acculab V-1200^®, IL, United States).