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Fluorescent Ca2+indicators have been essential for the analysis of Ca2+signaling events in various cell types. We showed that chemical Ca2+indicators, but not a genetically encoded Ca2+indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+chelating activity. Loading of commonly used Ca2+indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+and ATP. These results suggest that a critical review of data obtained with chemical Ca2+indicators may be necessary.

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Fluorescent dyes and probes are wonderful things, and they have been absolutely crucial to our understanding of cellular biology. Being able to see specific protein types and cellular structures in real time through a microscope with dyes, being able to monitor...