Fluorochromes in Flow Cytometry

A fluorescent compound has the property of absorbing light energy at a range of specific wavelengths. This absorption of light causes electron to rise from the ground state to a higher energy level (excited state). The excited electron quickly decays to its ground state while releasing the excess energy in the form of photon of light. This transition of energy is called fluorescence.

Cells are typically stained with fluorochromes to show the presence of components that otherwise would not be visible. The fluorochromes are usually fluorescence-conjugated antibodies. When a fluorescent dye is conjugated to a monoclonal antibody, it can be used to identify a particular cell type based on the individual antigenic markers of the cell. In a mixed cell population, different fluorochromes can be used to distinguish different subpopulations. The staining pattern of each subpopulation, combined with FSC and SSC data, can be used to identify which cells are present in the sample and to count their relative percentages. The cells can also be sorted if required.

The most widely used fluorochromes for labeling antibodies include FITC, PE and APC. Choosing the most suitable fluorochrome is an important issue, depending on the laser to be used. There are many fluorescent molecules with potential applications in flow cytometry. With the constant addition of new fluoresceins, the list is ever growing and we won’t cover all of them here.

Table 1. Partial flurochromes involved in flow cytometry.

Single Dyes

FITC, PE, APC and PerCP are single dyes that have been around for many years, but there are now alternatives that provide users with greater photo-stability and brighter fluorescence. In addition, alternative laser lines are becoming more affordable, so dyes excited by 355 nm and 405 nm lasers are increasing the choices for multiplexing.

Tandem Dyes

Tandem dyes involve the covalent coupling of a small fluorophore to another fluorophore. When the first dye is excited and reaches its maximal excited electronic singlet state, its energy is transferred to the second dye. This activates the second fluorophore and then produces a fluorescence emission. The process is known as Foster Resonance Energy Transfer (FRET). It is a good way to achieve a higher Stokes Shift, which increases the number of colors that can be analyzed from a single laser wavelength. Tandem dyes are very useful in multicolor fluorescence studies, especially in combination with a single dye.

Fluorescent Proteins

Fluorescent proteins, such as green fluorescent protein (GFP), mCherry and yellow fluorescent protein are also widely used for flow cytometry analysis and cell sorting. Fluorescent proteins are often co-expressed or fused with the protein of interest. The advantage of these fluorescent proteins is that they can quantitatively measure intracellular markers in living cells without the need to permeabilize the cell membrane.

References

1. Houston J P. et al.; Overview of fluorescence lifetime measurements in flow cytometry. Methods Mol Biol, 2018, 1678: 421-446.
2. Aysun Adan, et al.; Flow cytometry: basic principles and applications. Critical Reviews in Biotechnology, 2017, 37(2): 163-176.

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