Abstract
Increases in cytoplasmic free Ca(2+) ions ([Ca(2+)]) are a critical signal in pancreatic islet beta-cells and are usually required for insulin secretion in response to glucose or other secretagogues. Changes in Ca(2+), monitored using high-speed imaging across individual or multiple planes of the islet, can be used to explore the functional networks of beta-cells required for the precise regulation of insulin secretion. These networks are composed of functionally distinct beta-cell subpopulations: first-responders, highly connected hubs, and leader beta-cells, which initiate, connect, and dictate the pattern of spatially organized Ca(2+) oscillations, respectively. Alterations in Ca(2+) coordination among beta-cells contribute to defective insulin secretion, which underlies all forms of diabetes mellitus. Here, we provide a detailed protocol to perform Ca(2+) imaging in isolated rodent islets, focusing on mouse islets expressing the genetic Ca(2+) sensor, GCaMP6. We provide a step-by-step guide to evaluate general parameters of islet Ca(2+)dynamics, coordination, connectivity, and identification of specific functional subpopulations. This approach can be applied to investigate the role of Ca(2+) dynamics and coordination in tissues where coordination is critical for normal function.