Measurement of ATP production by islets depolarized with 70 mM KCl: islet ATP content and its accumulation in the extracellular medium in response to addition of the indicated glycolytic intermediate and its corresponding cofactors. Effects of the complex respiratory inhibitors 10 μM rotenone and 10 μM antimycin A.

Three groups, each of 25 islets, were incubated at 37°C for 60 minutes in 25 μl of KRBH. At the end of incubation, medium samples were separated and stored and islets consistently washed to dilute the contaminating extracellular nucleotide. After addition of perchloric acid to medium and islet samples, they were neutralized and potassium perchlorate precipitated. ATP was measured in samples of the clean supernatant with the luciferin/luciferase system. The emitted light was measured in a microplate reader (Synergy-2, Biotek) after addition of luciferase. More detailed information can be found in the Materials and Methods section.