Here, we describe SPoRTS, an open-source workflow for high-throughput spatiotemporal image analysis of fluorescence-based ratiometric biosensors in living spheroids. To achieve this, we have implemented a fully automated algorithm for the acquisition of line intensity profile data, ultimately enabling semi-quantitative measurement of biosensor activity as a function of distance from the center of the spheroid. We demonstrate the functionality of SPoRTS via spatial analysis of live spheroids expressing a ratiometric biosensor based on the fluorescent, ubiquitin-based cell-cycle indicator (FUCCI) system, which identifies mitotic cells. We compare this FUCCI-based SPoRTS analysis with spatially quantified immunostaining for proliferation markers, finding that the results are strongly correlated.
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| http://dx.doi.org/10.1016/j.crmeth.2025.100987 | DOI Listing |
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