Supplementary file 1_Pancreatic islet cell calcium ion imaging at single-cell resolution: functional identification of first-responder, highly connected (“hub”), and leader beta-cells.docx

Increases in cytoplasmic free Ca2+ ions ([Ca2+]) 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 Ca2+, 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 Ca2+ oscillations, respectively. Alterations in Ca2+ coordination among beta-cells contribute to defective insulin secretion, which underlies all forms of diabetes mellitus. Here, we provide a detailed protocol to perform Ca2+ imaging in isolated rodent islets, focusing on mouse islets expressing the genetic Ca2+ sensor, GCaMP6. We provide a step-by-step guide to evaluate general parameters of islet Ca2+dynamics, coordination, connectivity, and identification of specific functional subpopulations. This approach can be applied to investigate the role of Ca2+ dynamics and coordination in tissues where coordination is critical for normal function.