Non-Ratiometric Calcium Dyes
Certain fluorescent dyes are able to bind calcium and when they do so there is a quantitative increase in fluorescence. This occurs after dye loading into the cell in which the acteyl-methyl ester (AM) of Fluo 3 is cleaved of the dye as it passes across the plasma membrane. The resulting free dye is then free to bind Ca2+ ions within the cell. When the cell is activated, excess free Fluo-3 dye then binds the free Ca2+ resulting in an increase in dye fluorescence, which is excited at 488nm and emits at 530 nm .
Cell proliferation can now be measured flow cytometrically by the use of numerous probes including, Carboxyfluorescein Diacetate, Succinimidyl Ester or CFSE, PHK26, and now Violet Cell Trace, enabling researchers to easily monitor the proliferation rate of cells transfected with GFP, the original CFSE being in the same channel as GFP. If violet laser diodes are not available, a Red HeNe diode can be used to measure cell proliferation by the use of Cell Trace Far Red DDAO-SE.