Imaging Calcium Dynamics in Pancreatic Cells: A Technique to Study Real-time Changes in Cytosolic Calcium Concentration in Pancreatic Islet

- Pancreatic islets are a cluster of hormone-producing pancreatic cells that play a crucial role in maintaining glucose homeostasis. Elevated glucose concentration triggers the closure of potassium ion channels, preventing potassium ion efflux, leading to membrane depolarization. In consequence, voltage-dependent calcium ion channels open, allowing the influx of calcium ions. This increased cytosolic calcium concentration stimulates the release of hormones like insulin. In real-time, to image cytosolic calcium dynamics, transfer a few islets loaded with suitable calcium indicator dye between the spacer walls of a preassembled fine mesh.

Position the mesh upside down on a coverslip set inside the imaging chamber of an inverted microscope. Place a coarse mesh and weight to immobilize the cells. Add imaging solution into the chamber. Next, set up a perfusion system to introduce glucose or a test hormone of interest. Within the cells, preloaded fluorescent calcium indicators report any increase in cytosolic calcium concentrations. Upon binding to calcium ions, the indicator molecules exhibit a substantial increase in fluorescence emission. In the following protocol, we will show the re