Fluorescent Dyes:
Introduction - Quantifying calcium - Definitions&References - Protocols&Examples

Protocols & Examples

Reference experiment: effect of ionomycin in HeLa cells loaded with Indo-1 AM

Ratiometric indicators, such as Indo-1, are a bit more tricky to adquiere and quantify than non-ratiometric ones. This experiment using ionomycin exemplifies the advantages of ratiometric indicators.

In this case, we used HeLa cells loaded with Indo-1 AM and provoked an increase in intracellular calcium level with ionomycin (extracellular medium was HBSS- supplemented with 1µM Ca2+). This is equivalent to the example shown for Fluo-4 AM.

Emission of free indicator was recorded between 460 and 550 nm (channel 2) and the bonded form was measured between 380 and 450 nm (channel 1). Excitation was performed at 351 nm and the ratio between both images was generated using Leica's software.

Cells were focused using transmited light to avoid long UV-exposition (UV light might be cytotoxic!), and each adquisition channel was adjusted indepently to obtain a good singal to noise ratio. With this settings, channel 2 intensity decrease while channel 1 intensity increases.


Ratio images usually look more noisy than the original ones. In the case of Indo-1, changes in fluorescence present a broad dynamic range (more than 300%) that makes easy to follow small changes in calcium concentration. This is not the case with other ratiometrics indicators such as cameleons.


* Ratio image. Adquisition was performed using a Leica SP2 confocal microscope.

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