Glp 1 active elisa kit

The GLP-1 (active) ELISA kit was purchased from Millipore, and insulin ELISA kit was from Mercodia. For quantitative determination of cAMP, a competitive enzyme immunoassay kit from R&D system was used. All immunoassays were performed according to manufacturers’ instructions.

NCI-H716 cells were treated for 1 h with ADG at the indicated concentration. Some experiments included a 30 min incubation with triamterene prior to ADG treatment. After this, the supernatants were collected and tested for GLP-1 activity using a GLP-1 active ELISA kit (Millipore Co., Billerica, MA, USA). Each assay was performed in duplicate for the indicated samples. To determine the direct effect of ADG on insulin secretion in vitro [23 (link)], Min-6 cells were seeded in 12-well plates containing 1 mL of DMEM for 24 h prior to the experiment.
A 30 min incubation with triamterene was a prerequisite in certain experiments to examine the role of TGR5 in ADG-induced insulin secretion. The Min-6 cells were also pretreated for 30 min with the inhibitor at the indicated concentrations or the vehicle control at the same volume in a KRBH buffer containing 15 mmol/L glucose. The cells were then incubated in ADG at the indicated concentration for 1 h. The supernatants were then collected and the insulin levels in the media were then determined using an insulin ELISA kit (Mercodia, Uppsala, Sweden) [40 (link)]. Additionally, each assay was carried out in duplicate.

GLP1 content was extracted from ileal and proximal colonic tissue by homogenization of 1 g of tissue in 5 mL of ethanol acid solution (1% HCl 12 M, 74% absolute ethanol, 25% H₂O) (Polytron 3100, Kinematica, 24,000 rpm, 2 s × 20 s). After 24 h at 4°C, samples were centrifuged (20 min, 2,000 g, 4°C) and supernatants diluted (1:1000 and 1:250 for ileum and colon, respectively). Intestinal and plasma GLP1 concentration was measured using a GLP1 active ELISA kit (Millipore), according to the manufacturer instructions.

Adult mouse ileal crypt cell cultures were derived from approximately 10 cm of ileum, as previously reported (16 (link)). In brief, isolated crypts were suspended in 10% FBS-DMEM (with 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.1% v/v ROCK Inhibitor [Y-27632, Sigma-Aldrich]) and crypts from 1 ileum were plated onto a 24-well plate coated with Corning Matrigel (ThermoFisher Scientific) and incubated at 37°C with 5% CO2.
GLP-1 secretion assays were conducted 24-hour after crypt plating, as reported (16 (link)). Briefly, the cells were washed and then treated with 50 µM forskolin/50 µM IBMX or media alone (control) for 2 hours. Four wells were tested for each treatment group to make n = 1 per mouse. GLP-1 concentrations in the media and cell lysates were measured using a GLP-1 (active) ELISA kit (Millipore). Secretion was expressed as a percentage of the media GLP-1 content over the total GLP-1 content (media + cells).