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Calcium flux

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 .

Organelle Calcium Dye

Rhod-2 is a mitochondrial specific non-ratiometric calcium dye. This dye is sub-optimally excited at 488 nm and emits at 575 nm. The organelle specific nature of this dye allows the study of calcium trafficking between organelles such the endoplasmic reticulum (ER) and mitochondria.

Ratiometric Calcium Dyes

Indo-1 is a ratiometric calcium dye that is excited at 350-360 nm and emits at 390 nm and 530 nm. The dye when bound to calcium emits at 390 nm (violet) and when unbound emits at 530 nm (green), the ratio of the violet over the green signals is calculated as the ratio which is displayed as a parameter over time. |Hence before activation the presence of excess dye within the cell population should give a predominately green signal and hence low ratio value. Upon activation the dye should change from green to violet and hence the Indo-1 ratio should increase over time.

Useful book link:- Flow Cytometry - Principles and Applications

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