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

Protocols & Examples

Example of incomplete loading/de-esterification

Though there was no variation in cell morphology during this example, an increase in fluorescence was observed after adding ionomycin (HeLa cells were loaded with Fluo-4 AM and an increase in intracellular [Ca2+] was induced with 2µM ionomycin in HBSS+).


This graph indicate that the protocol used for loading cells has to be modified (different incubation conditions have to be tested in order to achieve an "ideal" behaviour: checking measurement conditions is also recomended).

Lower temperatures/less time in loading and de-esterification steps can produce an incomplete de-esterification. This problem causes an increase in the concentration of the free indicator during the experiment, which is reflected as a continual increase in fluorescence intensity. An insufficient de-esterification time can also produce variations in the fluorescence during the experiment (measurements are done at 37ºC, so samples incubated at room temperature need a time to equilibrate). Variations in cell morphology can also change mean fluorescence intensity.


Fluorescent Proteins


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