Fig. 5: Quantitative calcium measurements with Tq-Ca-FLITS in primary endothelial cells and human organoids.

a Calcium levels in ECs with plasma membrane targeted Tq-Ca-FLITS before and after stimulation with 1 µM histamine, indicated by an arrow. The color scale indicates the concentration of calcium and the fluorescence intensity. Scale bar is 40 µm, a representative of 17 cells is shown. b Time trace of the regions indicated in a. c–e Calcium concentration in ECs during TEM. The ECs express plasma membrane targeted Tq-Ca-FLITS and the leukocytes are labeled with Calcein Red-Orange. In total, 16 TEM events were captured within seven measurements, using four batches of neutrophils. c Representative fluorescence image of the ECs with the biosensor. The blue outline indicates the location of a neutrophil that crosses the EC monolayer. Scale bar is 30 µm. d Top panel, calcium concentration in ECs measured at the location of the neutrophil indicated in c and bottom panel, the fluorescence intensity of Tq-Ca-FLITS and the leukocyte at the same location. The intensity of the neutrophil drops right after crossing the EC monolayer, whereas the intensity of Tq-Ca-FLITS increases. The concentration of calcium in the EC remains constant, as determined from the fluorescence lifetime of Tq-Ca-FLITS. e Intensity fold-change (dF/F₀) of Tq-Ca-FLITS (top panel) and measured calcium concentration (bottom panel) in ECs before neutrophil contact (n = 216), during crawling of the neutrophil (n = 98), during diapedesis (n = 19) and post-diapedesis (n = 278). The boxplot indicates the median, the 95% CI (notches), the first and third quartiles (hinges), and the 1.5× interquartile range (whiskers). All data points are also indicated by circles. f–g Calcium changes measured in cells of a human small intestinal organoid stimulated with 10 μg/ml GPBAR-A. f A human small intestinal organoid expressing Tq-Ca-FLITS in the nuclei of the cells. Scale bar is 30 µm. Three organoids showed transient calcium increases, shown is a representative organoid. g Heatmap of time traces of the calcium concentration of the cells in f, without (left panel) and with (right panel) stimulation by GPBAR-A, indicated by an arrow. Markers indicate the corresponding cells in f. Source data are provided as a Source Data file.