Abstract
Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.
Highlights
Transient, follow many different pathways, and can vary significantly from cell to cell
To overcome these limitations and to facilitate the automated quantification of global intracellular Ca2+ responses we developed a bespoke MATLAB-based software for Calcium Quantification (CalQuo)
We exemplified our approach by examining the calcium responses of T-cells settling on functionalized microscope cover glass
Summary
The use of CalQuo in combination with fast spinning-disc microscopy and fluorescent calcium reporters allowed the quantification of the number (or fraction) of calcium-releasing cells, including the time-dependent global calcium responses, and the interval between stimulation and calcium release. At present the software requires a minimal level of MATLAB-based operating knowledge. CalQuo is well suited to studying other cell signaling processes, such as the release of proteins or other molecules (e.g. toxins), if these molecules can be detected using fluorescent labels. We expect CalQuo to contribute to a more facile and better understanding of the molecular basis of global signaling in eukaryotic cells
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