Dual Detection System for Simultaneous Measurement of Intracellular Fluorescent Markers and Cellular Secretion

Glucose-stimulated insulin secretion from pancreatic β-cells within islets of Langerhans plays a critical role in maintaining glucose homeostasis. Although this process is essential for maintaining euglycemia, the underlying intracellular mechanisms that control it are still unclear. To allow simultaneous correlation between intracellular signal transduction events and extracellular secretion, an analytical system was developed that integrates fluorescence imaging of intracellular probes with high-speed automated insulin immunoassays. As a demonstration of the system, intracellular [Ca²⁺] ([Ca²⁺]_i) was measured by imaging Fura-2 fluorescence simultaneously with insulin secretion from islets exposed to elevated glucose levels. Both [Ca²⁺]_i and insulin were oscillatory during application of 10 mM glucose with temporal and quantitative profiles similar to what has been observed elsewhere. In previous work, sinusoidal glucose levels have been used to test the entrainment of islets while monitoring either [Ca²⁺]_i or insulin levels; using this newly developed system, we show unambiguously that oscillations of both [Ca²⁺]_i and insulin release are entrained to oscillatory glucose levels and that the temporal correlation of these are maintained throughout the experiment. It is expected that the developed analytical system can be expanded to investigate a number of other intracellular messengers in islets or other stimulus-secretion pathways in different cells.