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)

Facs Fluorochromes

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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Responses

  • KALIMAC SACKVILLE
    Which dye can be excited by 488?
    6 years ago
  • idris
    When happens when a fluorochrome is excited by an energy source during flow cytometry?
    6 years ago
  • Pervinca
    What is the most number of fluorochromes that can be used on one cytometer?
    6 years ago
  • MARTINA
    What are tandem fluorochromes?
    6 years ago
  • ILARIA
    Which fluorochromes cannot be used together in flow cytometry?
    6 years ago

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