Fig. 2

Optimization of conditions to measure IP1 accumulation in dispersed islets. a MIN6 cells were seeded into 96-well plates and incubated with 100 μM CCh with or without the Gq/11 inhibitor FR900359. CCh induced a significant increase in IP1 concentration which was completely abolished by FR900359. b Dispersed islets were seeded into coated 96-well plates to become adherent overnight and incubated with 100 μM CCh for 4 h, however, CCh failed to induce IP1 accumulation. c Using a suspension-based protocol for IP1 determination, dispersed islets were exposed immediately after dispersion to 100 μM CCh for 4 h. CCh also failed to raise IP1 levels in primary islet cells. d Reducing the incubation time to 1 h led to a significant increase of IP1 in CCh treated cells. This effect was dependent on Gq/11 activity because application of the Gq/11 inhibitor FR900359 completely abolished IP1 accumulation. Data are given as means ± SEM of three to five independent experiments each performed in triplicates. Statistical significance was tested with the two-tailed paired t test (**p ≤ 0.01; ***p ≤ 0.001)