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

Introduction - Choosing a dye - Loading

Fluorescent Dyes - Loading

Calcium indicators are unable to cross lipid membranes due to their nature, making necessary the use of physical or chemical methods to load them inside the cell.

Loading Acetoxymethyl (AM) esters: the protection of carboxylic groups as AM esters makes the dye neutral, so it can cross the cell membrane. Once inside the cell, esterases will cleave AM groups. This process gives place to charged compounds that are entrapped inside the cell. A scheme of this process is represented below (red balls represent AM protecting groups, while the big ball represents the fluorescent dye core).

 

Complete hydrolysis of the AM esters is very important to avoid artefacts. If the experiment begins before all AM ester dye is converted to free dye, total concentration increases during the experiment and gives place to false fluorescence variations (see an example).

Complete hydrolysis is still more crucial if the AM ester is already fluorescent. This is the case of Indo-1 AM and Fura-2 AM, which are fluorescent and calcium insensitive. If de-esterification is not completed, there will be a mixture of indicator (free and bounded to calcium) and indicator-AM. When the three forms of the indicator are fluorescent, the signal depends not only on calcium levels, but also on the non de-esterificated indicator concentration.

An example of a protocol used for loading AM esters in HeLa cells is available for Fluo-4 AM and Indo-1 AM.

Though charged indicators cannot cross cell membrane, intracellular concentration might decrease during experiment due to photobleaching and/or active transport out of the cell.

A drawback of AM esters is that they can accumulate inside intracellular compartments, making indicator insensitive to calcium cytosolic levels. To avoid this behaviour, loading/de-esterification temperature can be decreased, which usually implies longer incubation periods.

Loading of dextran conjugates and salt form: these compounds are impermeable to cell membranes, so they are less prone to accumulate in intracellular compartments. As a drawback, they have to be load using reversible methods to make permeable large populations or procedures such as microinjection and electroporation (cells can be damaged during these processes).

An important advantage of dextran conjugates is that dextran moiety not only avoids compartmentalization, but also prevents conjugation of the indicator to proteins, membranes, etc. This prevents indicator from being sequestered and provides enhanced resistance to leakage.

 

Protocols for:

 

Fluorescent Proteins

 

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