Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry |
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Authors: | Wing-Kee Lee Thomas Dittmar |
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Affiliation: | 1Institute for Physiology, Pathophysiology, & Toxicology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke;2Institute for Immunology & Experimental Oncology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke |
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Abstract: | A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin. |
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Keywords: | Cellular Biology Issue 92 Kidney FACS second messenger proximal tubule calcium indicators probenecid endoplasmic reticulum ionomycin |
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