Rat insulin elisa kit

A rat insulin ELISA kit (Gentaur, Shibayagi Co. Ltd., Gunma, Shibukaw, Japan) was used to determine the insulin secretory function of PRPE and compounds 1–15 on INS-1 cells. INS-1 cells were seeded at a density of 400,000 cells per well in 12-well plates for 24 h. Afterwards, INS-1 cells were first incubated in a Krebs–Ringer bicarbonate buffer (KRB, pH 7.4) and starved for 1 h. The KRB was then removed and preincubated with PRPE, compounds 1–15, or gliclazide for 30 min, then stimulated for 1 h with KBR with low glucose (3.3 mM) or high glucose (16.7 mM), respectively, as an INS-1 cell stimulant. The KRB was collected, and the insulin concentration in KRB was measured using a rat insulin ELISA kit in accordance with the manufacturer’s instructions. The optical density at 450 nm was measured using a microplate reader (PowerWave XS, Bio-Tek Instruments, Winooski, VT, USA). The glucose stimulation index (GSI) was calculated as follows: GSI = insulin level in high glucose (16.7 mM)/insulin level in low glucose (3.3 mM).

Glucose-stimulated insulin secretion (GSIS) was evaluated using a rat insulin ELISA kit (Gentaur, Shibayagi Co. Ltd., Gunma, Shibukaw, Japan). INS-1 cells were seeded (5 × 10⁵ cells/well) in 12-well plates. After incubation for 24 h, each well was washed twice with Krebs-Ringer bicarbonate HEPES buffer (KRBB, 4.8 mM KCl, 129 mM NaCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 10 mM HEPES, 5 mM NaHCO3, and 0.1% BSA, pH 7.4) and 2.8 mM glucose. Before treatment, the cells were allowed to starve in fresh KRBB. After incubation for 2 h, the cells were treated with KRBB containing test samples and gliclazide (positive control), and then KRBB containing basal (2.8 mM) and stimulating (16.7 mM) glucose concentrations was added to each well. After incubation for 1 h, the supernatants from each well were collected and centrifuged at 12,000 rpm and 4 °C for 10 min and then GSIS was assessed using a rat insulin ELISA kit according to the manufacturer’s instructions. The glucose stimulation index (GSI) was calculated by dividing the insulin level at the stimulating (16.7 mM) glucose concentration by the insulin level at the basal (2.8 mM) glucose concentration and was compared with the control (untreated glucose-stimulated cells).

GSIS was measured using a rat insulin ELISA kit (Gentaur, Shibayagi Co. Ltd., Gunma, Shibukaw, Japan) in accordance with the manufacturer’s instructions. INS-1 cells were incubated with Krebs–Ringer bicarbonate HEPES buffer (KRBB; 4.8 mM KCl, 129 mM NaCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 10 mM HEPES, 5 mM NaHCO₃, and 0.1% BSA, pH 7.4) containing α-Spinasterol, gliclazide (positive control), nifedipine (L-type Ca²⁺ channel blocker), Bay K 8644 (L-type Ca²⁺ channel activator), diazoxide (K⁺ channel activator), or glibenclamide (K⁺ channel blocker) in 12-well plates for 2 h; this was followed by incubation in KRBB containing 2.8 mM and 16.7 mM glucose for 1 h. After termination of treatment, GSIS was assessed using a rat insulin ELISA kit in accordance with the supplier’s instructions.