Intracellular Ca++ fluxes were visualized by the Ca++-sensitive dye fluo-4 AM (Invitrogen) following the manufacturer’s recommendations. Briefly, host cells were incubated for 30 min in fluo-4 AM (2.5-μM final concentration) at 37 °C and supplemented with pluronic acid (Invitrogen) in a dye/pluronic acid ratio of 1:1. Non-incorporated dye was removed by washing in sterile PBS and adding fresh modECGM. Calcium influx induction was induced by calcium ionophore A23187 treatments (15 μM, Sigma-Aldrich).
Post-processing analysis was performed by the use of Image J v1.52p software (Schneider et al. 2012 (link)). Z Project plugin was applied to holotomographic z-stacks with the average intensity of the images as projection output. For calcium flux measurements over time, defined areas surrounding resting host cells were defined as regions of interest (ROI) (Silvestre-Roig et al. 2019 (link)). Thereafter, the multi-measurement tool (roiManager “Multi Measure”) was used to quantify the mean grey value as indicator of fluorescence intensity over time. Finally, for better visualization of Ca++ flux, fluorescence images were displayed in pseudo-colours, using fire lookup tables as described elsewhere (Ardiel et al. 2017 (link); Liu and Baraban 2019 (link); Wakida et al. 2020 (link)).
Post-processing analysis was performed by the use of Image J v1.52p software (Schneider et al. 2012 (link)). Z Project plugin was applied to holotomographic z-stacks with the average intensity of the images as projection output. For calcium flux measurements over time, defined areas surrounding resting host cells were defined as regions of interest (ROI) (Silvestre-Roig et al. 2019 (link)). Thereafter, the multi-measurement tool (roiManager “Multi Measure”) was used to quantify the mean grey value as indicator of fluorescence intensity over time. Finally, for better visualization of Ca++ flux, fluorescence images were displayed in pseudo-colours, using fire lookup tables as described elsewhere (Ardiel et al. 2017 (link); Liu and Baraban 2019 (link); Wakida et al. 2020 (link)).
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