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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