Which fluorochromes are useful for flow cytometry?
There are dozens of fluorescent molecules (fluorochromes) with a potential application in flow cytometry. The list is ever growing but it is not the scope of this guide to cover them all. Instead, some of the most useful fluorochromes for surface or intracellular epitope detection are described below, including the very latest in fluorescent probe technology – tandem dyes. There is enough variation in the two tables to cover most researchers’ needs.
Single dyes:
Some of these single dyes e.g. FITC have been in use for the past 30 years but are now facing competition from alternatives like Alexa Fluor® dyes, which offer the user greater photostability and increased fluorescence.
Tandem dyes:
In a tandem dye, a small fluorochrome takes a ‘piggy-back’ ride on another larger fluorochrome. When the first dye is excited and reaches its maximal singlet state, all its energy transfers to the second dye (an acceptor molecule), located in close proximity. This activates the second fluorochrome, which then produces the fluorescence emission. The process is called FRET (fluorescence resonance energy transfer). It is a clever way to achieve higher Stokes Shifts and, therefore, increase the number of colors that can be analyzed from a single laser wavelength.
The majority of tandem dyes have been manufactured for the standard 488 nm laser, which is found in most flow cytometers. Tandem dyes are very useful for multicolor fluorescence studies especially in combination with single dyes. For example, Alexa Fluor® 488, Phycoerythrin, PerCP-Cy5.5 and PE-Cy7 can all be excited at 488 nm, but will produce green, yellow, purple and infrared emissions respectively, which can be measured using separate detectors.
Note. Phycoerythrin (PE) is same as R-Phycoerythrin (RPE).
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