Simultaneous monitoring of Zn~(2+) secretion and intracellular Ca~(2+) from islets and islet cells by fluorescence microscopy

摘要

A methodfor simultaneously imaging Zn~(2+) secretion and intracellular Ca~(2+) at beta-cell clusters and tingle islets of Langerhans was developed. Cells were loaded with the Ca~(2+) indicator Fura Red, incubated in buffer containing the Zn~(2+) indicator FluoZin-3, and imaged via law scanning fluorescence confocal. microscopy. FluoZin-3 and Fura Red we excited at 488 nm and emit at 515 and 665 nm respectively Zn~(2+), which is co-released with insulin, reacts with extracellular FluoZin-3 to form a fluorescent product Stimulation of cell clusters with glucose evoked increases and oscillations in intracellular Ca~(2+) and Zn~(2+) secretion that were correlated with each other and. were synchronized among cells. In single islets, s-patially resolved dynamics of secretion including detection of first phase, second phase, and synchronized oscillations around the islet were observed Fura Red did not yield detectable Ca~(2+) signals at islets For islet measurements, cells were loaded with Fura-2 and incubated in FluoZin-3 while sequentially-illuminating the islets with 340 380 and 470 nm light and acquiring epi-fluorescence images with a charge-coupled device (CCD) camera. This allowed Ca~(2+) and secretion to be observed with approximately 2 s temporalresolution. This method should be useful for studying Ca~(2+) secretion, coupling and any application requiring rapid assays of secretion.