Measuring ARPE19 Intracellular Calcium Levels

In the images of ARPE19 that were stained with 1 µM Fluo 3/AM (at dark for 60 min) (ThermoFisher Sci.), we measured the cCa²⁺ concertation [16 (link), 25 (link)]. Axio Observer 7 inverted microscope with 20 × objective (Carl-Zeiss) was attached to the LSM-800 setup for the laser stimulation of the Fluo 3/AM in the labeled cells. The extracellular solution was applied for removing the Fluo 3/AM dye from the cells prior to adding the TRPM2 stimulator (1 mM and H₂O₂) to the glass bottom dishes. For antagonizing the TRPM2 channel, 200 µM CRV was used in the LSM-800 experiments of Fluo 3/AM [22 (link)]. A specific computer with high resolution was used to measure the changes in the fluorescence intensity in the images taken by ARPE19 using a specific ZEN application (version ZEN 3.2 blue edition and Carl-Zeiss). The fluorescence intensity result was expressed using the a.u.