78 Steps Health Journal

Fluorochromes For Flow Cytometry

A wide variety of fluorochromes have been used in flow cytometry. The number of fluorochromes still keep on growing, as new applications of flow cytometry dictate the need for novel fluorochromes to be developed. The choice of fluorochromes is primarily dictated by specific applications as well as by the laser excitation source available on the flow cytometer. For example, for phenotyping, fluorochromes have been preferred because they can easily be conjugated to antibodies and are excitable by the popular argon ion laser.

Table 11.4 lists many of the fluorochromes and their common abbreviations, if any, used in flow cytometry. It also lists the excitation and emission wavelengths, along with their typical applications. For most applications, it is desirable to use more than one fluorchrome so that one can conduct multiparameter analysis of the specimen. For this application, it is desirable to have a flow cytometer with more than one laser excitation source to offer a wide choice in selecting the fluorochromes. However, in the case where the flow cytometer has only one laser such as an argon ion laser providing 488-nm excitation, one can select a dye pair such that each dye has a different amount of Stokes shift (separation between the excitation wavelength and the emission peaks as discussed in Chapter 4). With such a pair, even though the excitation is provided at the same wavelength, the emission spectra from the two dyes are well separated in two different regions. Figure 11.7 represents the excitation and emission spectra of some fluorochromes.

The two most commonly used fluorochromes for dual color flow cytometry are (i) fluorescein, often abbreviated as FITC because it is the fluorescein isothiocyanate form that is used for conjugation with specific antibodies for phenotyping application, and (ii) phycoerythrin, abbreviated as PE, which is derived from red sea algae. As can be seen from Figure 11.7, both these dyes can be excited at 488 nm. However, their fluorescence peaks are well separated; while FITC emits in the green (~520nm), the emission from PE is of orange color (~575nm).

Another approach used to separate the emissions of two fluorochromes is that of a tandem fluorochrome. Here one utilizes a combination of two dyes, one absorbing efficiently at the excitation wavelength and then exciting another chemically attached dye by Förster energy transfer, which then emits at a wavelength considerably red shifted. An example of a natural tandem dye is PerCP (peridinin chlorophyll protein), which is a carotenoid:chlorophyll complex. Here the carotenoid unit absorbs at 488 nm and transfers energy to chlorophyll, which emits at ~670nm. Hence the tandem dye exhibits a large apparent Stokes shift. The excitation and the fluorescence spectra of this tandem fluorochrome are also shown in Figure 11.7. A synthetic tandem fluorochrome is PE covalently linked to Cy5 whose excitation and florescence spectra are also shown in Figure 11.7. It can be excited at 488nm by absorption into the PE unit which transfers energy to Cy5 emitting at 670nm. For a

TABLE 11.4. List of Fluorochromes for Flow Cytometry

Fluorochrome	Excitation	Emission	Applications
	(nm)	(nm)
Fluorescein (FITC)	495	520	Phenotyping
R-Phycoerythrin (PE)	480	575	Phenotyping
Tricolor	488	650	Phenotyping
PerCP	470	670	Phenotyping
TRITC (Tetramethyl	488	580	Phenotyping
rhodamine)
Coumarin	357	460	Phenotyping
Allophycocyanin (APC)	650	660	Phenotyping
APCCy7	647	774	Phenotyping
Cascade blue	350	480	Phenotyping
Red 613	480	613	Phenotyping
Texas red	595	620	Phenotyping
Cy3	550	570	Phenotyping
Cy5	648	670	Phenotyping
Red 670	480	670	Phenotyping
Quantum red	480	670	Phenotyping
Hoechst 33342	350	470	DNA analysis/apoptosis
Hoechst 33258	350	475	DNA analysis/chromosome
			staining
DAPI	359	462	DNA staining, preferentially of
			AT sequences
Chromomycin A3	457	600	DNA analysis/chromosome
			staining
Propidium iodide	495	637	DNA analysis
Ethidium bromide	518	605	DNA analysis
TOPRO3	642	661	DNA analysis
Acridine orange	490	530/640	DNA, RNA staining
Sytox green	488	530	DNA
Fluorescein diacetate	488	530	Live/dead discrimination
SNARF1	488	530/640	pH measurement
Indo1	335	405/490	Calcium flux measurement
Fluo3	488	530	Calcium flux measurement
Rhodamine 123	515	525	Mitochondria staining
Monochlorobimane	380	461	Glutathione specific probe

Source: http//www.icnet.uk/axp/facs/davies/Flow.html.

Source: http//www.icnet.uk/axp/facs/davies/Flow.html.

three-color analysis, using 488 nm, the PECy5 tandem together with FITC and PE have been the frequent choice for phenotyping application.

A number of fluorochromes have been used for nucleic acid staining and analysis of DNA content. These fluorochromes have been described in Chapter 8. The most popular choice as a DNA fluorochrome for flow analysis is propidium iodide. This fluorochrome is not very specific because it stains

Excitation

Emission

400 500 600 700 800

Marina Blue

Cascade Blue®

Cascade Yellow

Fluorescein

Phycoerythrin

Texas Red®

Allophycocyanin

PharRedTM (Cy7-APC)

400 500 600 700 800

Marina Blue

Cascade Blue®

Cascade Yellow

Fluorescein

Phycoerythrin

Texas Red®

Allophycocyanin

PharRedTM (Cy7-APC)

400 500 600 700 800

Wavelength (nm)

Figure 11.7. Excitation and emission (bold) spectra of some fluorochromes used in flow cytometry. (Reproduced with permission from BD Biosciences.)

400 500 600 700 800

Wavelength (nm)

Figure 11.7. Excitation and emission (bold) spectra of some fluorochromes used in flow cytometry. (Reproduced with permission from BD Biosciences.)

the double-stranded regions of both DNA and RNA. Also, it is not able to penetrate an intact cell membrane (live cell). However, propidium iodide has the advantage of being efficiently excitable by the 488-nm line of an argon ion laser, whereas other DNA dyes such a DAPI, Hoechst and acridine orange require different wavelengths for efficient excitation. Table 11.4 lists the excitation wavelengths for these dyes. Both DAPI, which specifically stains the AT base pair, and Hoeshst, which stains the GC base pair on DNA, requires excitation in the UV. As discussed in Chapter 8, acridine orange shows an interesting feature in that it fluoresces red when bound to single-stranded or nonhelical nucleic acid such as RNA or denatured DNA, but fluoresces green when intercalated in the double-stranded helical nucleic acid of native DNA. This change in fluorescence color can be used to study the denaturability of DNA during the cell cycle.

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