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

Protocols & Examples

Reference experiment: effect of ionomycin in HeLa cells loaded with Fluo-4 AM

To check system response and adjust experimental conditions, it is a good idea to use a ionophore to get a reference experiment.

We used HeLa cells loaded with Fluo-4 AM for this example and provoked an increase in intracellular calcium level with ionomycin (extracellular medium was HBSS- supplemented with 5µM Ca2+).

A region with some cells homogeneous labelled is chosen and fluorescence is traced every 5 seconds (higher speed is not needed in this case). Some frames are recorded to define initial state and then ionomycin is added to increase intracellular calcium concentration.


It is easy to appreciate in the picture that every cell has a starting fluorescence intensity slightly different. There is still more difference in final fluorescence: a more detailed graphical analysis illustrates this behaviour.


* Image was acquired using a Leica SP2 confocal microscope.

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