I also want to use Spectro fluorometer to measure calcium concentration. One more thing, after treating cells with calcium dye, Should I lyse the cells or not to measure calcium concentration?
Thanks in Advance
The purpose of using the AM ester of Fluo-8 and other intracellular calcium indicators is to mask the carboxylate charges so that the dye can get into the cells. Once it is in there, esterases inside the cell cut off the AM ester, leaving the active dye. If you want to measure calcium concentration in solution using the dye, you should use the free acid form, not the AM ester.You will need to make a set of standards to calibrate the fluorescence versus [Ca2+] response.
If you want to measure the calcium concentration inside the intact cells, consider using a ratiometric dye, in which the ratio of fluorescence measured at 2 different wavelength settings reports on the calcium concentration. Then you don't have to worry about lysing the cells to get the calcium out.
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/ion-indicators/calcium-indicators.html
https://www.abcam.com/ps/products/112/ab112129/documents/Fluo-8-No-Wash-Calcium-Assay-Kit-v3b-ab112129%20(website).pdf
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