4.6. Glucose-Stimulated Insulin Secretion (GSIS) Assay

The experiment began by seeding β-cells in a 24-well plate, with each well initially containing 0.5 × 106 cells. These cells were then left to incubate for a period of 2 days. Following the incubation, the cells were subjected to a 2-h pretreatment using media containing either eugenol or a control vehicle (ethanol). After this pretreatment, the cells were exposed to HG-HL conditions for a total duration of 48 h.

To assess glucose-stimulated insulin secretion (GSIS), a specialized solution was prepared using HBSS with precise salt concentrations and the addition of bovine serum albumin, all adjusted to a pH of 7.2. The cells were then thoroughly washed with HBSS. During the second wash, which extended for 1 h, any residual substances were removed. The experimental groups were distributed across two wells. One well was treated with HBSS containing 10 μM eugenol and 2.5 mM glucose, representing fasting blood glucose in rats. In contrast, the other well received HBSS with 10 μM eugenol and 16.5 mM glucose, representing postprandial blood glucose. Both sets of wells were subsequently incubated for 2 h, and after this incubation period, the solutions were collected for analysis via insulin ELISA.

We utilized the Rat/Mouse Insulin ELISA kit (EZRMI-13K) from Sigma Aldrich (Oakville, ON, Canada) for our ELISA analysis. To prepare for the ELISA, we placed the ELISA strips into a holder, and each well underwent a triple rinse with 300 μL of diluted Wash Buffer. Following the rinsing, we introduced 10 μL of each sample into the wells, and this was succeeded by the addition of 80 μL of the Detection Antibody. The entire plate was sealed and then incubated at room temperature on an orbital microtiter plate shaker. After incubation, we removed the sealer and carefully emptied the solutions from the wells. The wells then experienced three additional washes with the diluted Wash Buffer and were gently tapped on a paper towel to eliminate any excess buffer. Next, we introduced 100 μL of Enzyme Solution into each well, resealed the plate, and incubated it with moderate shaking for 30 min. Once this step was completed, we performed six more washes with the diluted Wash Buffer. Subsequently, 100 μL of Substrate Solution was dispensed into each well. The plate was sealed once more and gently shaken for a period of 5–20 min. After removing the sealer, we added 100 μL of Stop Solution to each well and briefly shook the plate to ensure thorough mixing. Finally, we measured the absorbance of the plate at 450 nm and 590 nm using SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).