HTRF measurement of dopamine and bromocriptine effects on insulin secretion in cells and islets.

<p><b>(A)</b> Increasing concentrations of dopamine (DA) caused dose-dependent inhibition of GSIS in INS-1E cells, which was best fit to a sigmoidal curve (IC₅₀ = 1.28 ± 0.06 μM, R² = 0.93). <b>(B)</b> Similarly, treatment of wildtype mouse islets with 10 μM DA significantly and comparably inhibited GSIS (p<0.001) by 70.7 ± 6.8%. Consistent with a role for dopaminergic signaling as a negative mediator of GSIS, treatment of islets with10 μM bromocriptine inhibited GSIS by 67.4 ± 8.1%. For INS-1E cell-based and mouse islet experiments (<b>Panels A and B</b>, respectively), data are represented as % maximal insulin secretion based on mean HTRF values ± SEM from n≥3 independent experiments. For all panels, HTRF measurements were performed in 96-well plates with INS-1E cell secretion experiments performed in triplicate and mouse islet experiments performed in hextuplicate.</p

Validation of HTRF insulin assay conditions.

<p><b>(A)</b> pH significantly affected the ratiometric HTRF signal across a range of human insulin concentrations [F(2, 40) = 21.35, p<0.001]. Antibody incubation at pH 7 yielded the greatest HTRF signal compared to other pHs (p<0.001). <b>(B)</b> 2 h antibody incubation was sufficient to produce a robust HTRF signal, with longer antibody incubation times (12 and 48 h) further increasing HTRF signal. <b>(C)</b> There was a temperature-dependent difference in HTRF values when antibodies were incubated for 2 h at room temperature (RT, 25°C) versus 37°C [F(1,29) = 16.57, p<0.001] with higher signal observed at RT. <b>(D)</b> There was no significant difference in HTRF signal between human, rodent, porcine and bovine insulin across a range of concentrations (0.01–10 nM; p>0.05). <b>Panels A-C:</b> Data are represented as the mean emitted HTRF ratio ± SEM. <b>Panel D:</b> Data are represented as %ΔF of the HTRF signal for the respective species. For all panels, the data are from experiments performed in triplicate in 384-well plates.</p

Advantages of the HTRF-based insulin assay compared to ELISA and RIA approaches.

<p>Here we show the main advantages of an HTRF insulin assay over comparable RIA and ELISA-based methods. Though all three methods are similarly sensitive and specific for insulin detection, the homogenous nature of the HTRF assay eliminates numerous reagents and mixing, washing and blocking steps, making the assay shorter, less expensive and more amenable to medium and high-throughput screens.</p

Comparison between HTRF and ELISA insulin detection assays.

<p>Supernatants collected from glucose-stimulated INS-1E cells (20 mM glucose, 90 min, 37°C) were measured concurrently with HTRF or ELISA insulin assays. The respective HTRF and ELISA assay-derived insulin concentration values were plotted. A linear regression curve of the data showed close correlation of the insulin values from the two methods (slope = 1.15 ± 0.16, R² = 0.84). Results are represented as mean insulin concentrations ± SEM performed in triplicate in 3 independent experiments.</p