title: Cytosolic calcium measurements in renal epithelial cells by flow cytometry.
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.
authors.affiliation: Institute for Physiology, Pathophysiology, & Toxicology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke; Wing-Kee.Lee@uni-wh.de.
authors.forename: Wing-Kee
authors.initials: WK
authors.lastname: Lee
authors.affiliation: Institute for Immunology & Experimental Oncology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dittmar
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D061207:Calcium Ionophores / Q000494:pharmacology; D002448:Cell Adhesion / Q000502:physiology; D004396:Coloring Agents / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D015759:Ionomycin / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007783:Lactones / Q000494:pharmacology; D051381:Rats; D012717:Sesquiterpenes / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D061207:Calcium Ionophores; D004396:Coloring Agents; D007783:Lactones; D012717:Sesquiterpenes; D014415:Tunicamycin; D015759:Ionomycin; C046319:thapsigargicin; D002118:Calcium