Protocols

Here we present the absorption and emission spectra of the fluorochromes BD Biosciences Pharmingen conjugates to monoclonal antibodies and other proteins. Many of these fluorochromes, including fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), PE-Cy5, and PerCP, can be used with single-laser flow cytometers equipped with an argon-ion laser emitting light at 488 nm for three-color flow cytometric analysis (Fig. 1). For details on the excitation and emission spectra of fluorochromes, see our interactive Fluorescence Spectrum Viewer at http://www.bdbiosciences.com/spectra/.

BD Biosciences Pharmingen also offers monoclonal antibodies and proteins conjugated to allophycocyanin (APC) and avidin conjugated to Texas Red™. Both of these dyes are useful in experiments where multi-color analysis is desired using flow cytometers with dual laser capabilities (Fig 1). APC can be excited by a helium-neon (HeNe) laser emitting light at 633 nm, by a krypton laser emitting light at 647 nm, or by a dye laser which can be conveniently tuned to emit light in the 550-650 nm range. (In a dye laser, the lasing medium is a solution of fluorescent dye excited by a pump laser, usually an ion laser.) Texas Red™ , available conjugated to avidin, can be excited by an argon-krypton mixed-gas laser at 568 nm, or with a dye laser, where both APC and Texas Red™ can be used simultaneously. Because many combinations of lasers, detectors, filters and fluorochromes are possible for multi-color analysis, proper precautions need to be taken (i.e.,bandpass filters, dichroic mirrors, longpass filters, etc.) by the operator to ensure each fluorochrome is being detected by only one detector (Fig. 1). In the following descriptions, we give our recommendations for the ideal instrument set-up for use with our reagents.(summarized in Table1)

Allophycocyanin (APC) is an accessory photosynthetic pigment found in bluegreen algae. Its molecular weight is approximately 105 kDa. APC has 6 phycocyanobilin chromophores per molecule, which are similar in structure to phycoerythrobilin, the chromophore in R-PE. It has a 650 nm wavelength absorption maximum (Fig. 2) and a 660 nm fluorescence emission maximum (Fig. 2). Using a 660 ± 10 nm BP filter will give optimum detection for this fluorochrome. APC can be used in flow cytometers equipped with dual lasers for multi-color analysis (Fig. 2). It can be excited by laser light between 600-640 nm. For this, we recommend a He-Ne laser at 633 nm, or a tunable dye laser tund between 600-640 nm.

PE-Cy5 is a tandem conjugate system, with an absorption maximum of approximately 650 nm (Fig. 2), which combines R-phycoerythrin and a cyanine dye (MW 1.5 kDa). This product was previously known as BD Cy-Chrome. When excited by 488-nm light, the excited fluorochrome (PE) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent maximum is approximately 670 nm (Fig. 2). Using a 650-nm longpass filter will give optimum detection for this fluorochrome. The efficiency of the light energy transfer between the two fluorochromes can be seen in Fig. 2F where less than 5% of the absorbed light is lost as fluorescence at 575 nm by PE. Compared to other fluorescence energy-transfer systems used in flow cytometry (e.g., RED613™ , EDC, PerCP), PE-Cy5 is a superior fluorochrome for third color analysis because of its high emission intensity and broad spectrum. As with our PE conjugates, an average of one PE-Cy5 molecule is coupled per antibody or protein. Because of its broad absorption range (Fig. 2), PE-Cy5 is not recommended for use with dual-laser flow cytometers where excitation by both lasers is possible.

Precautions for flow cytometry: PE-Cy5 tandem fluorochromes have been reported to bind some classes of human macrophages and granulocytes via Fc receptors, and PE has been reported to bind to mouse B lymphocytes via Fc receptors. Preincubation of mouse leukocytes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 can reduce the non-specific binding of PE-Cy5-conjugated reagents to mouse B cells. However, PE-Cy5 conjugated reagents should not be used to stain splenocytes of SJL, NOD, and MRL mice as B lymphocytes and/or other leukocytes have been reported to non-specifically stain regardless of the use of Mouse BD Fc Block™. Reagents conjugated to PE, PerCP, PerCP-Cy5.5, APC, and APC-Cy7 tandem fluorochrome can be used on leukocytes from these mouse strains.

Fluorescein isothiocyanate (FITC) is a fluorochrome with a molecular weight of 389 daltons and an absorption maximum at 495 nm (Fig. 2). Its excitation by 488-nm light leads to a fluorescence emission maximum around 520 nm (Fig. 2). Using a 530 ± 15 nm bandpass (BP) filter will give optimum detection for this fluorochrome. The isothiocyanate derivative (FITC) is the most widely used form for conjugation to antibodies and proteins, but other derivatives are available. FITC has a high quantum yield (efficiency of energy transfer from absorption to emission fluorescence) and approximately half of the absorbed photons are emitted as fluorescent light. The number of FITC molecules per conjugate partner (antibody, Avidin, Streptavidin, etc.) is usually in the range of three to five molecules.

R-phycoerythrin (PE) is an accessory photosynthetic pigment found in red algae. In vivo, it functions to transfer light energy to chlorophyll during photosynthesis. In vitro, it is a 240-kDa protein with 34 phycoerythrobilin fluorochromes per molecule. The large number of fluorochromes per PE molecule make it an ideal pigment for flow cytometry applications. Its absorption maximum is 564 nm (Fig. 2). When excited by 488-nm light, its fluorescence emission maximum is approximately 575 nm (Fig. 2). For single-laser flow cytometer use, we recommend using a 585 ± 21 nm BP filter for optimal detection (Fig. 1). When performing multi-color analysis with a dual-laser system, a tighter window of detection is required to compensate for the other conjugates being used (e.g.,Texas Red ™ ). For this, we recommend using a 575 ± 13-nm BP filter (Fig.1). Our conjugation chemistry yields an average of one PE molecule per antibody or protein. The emitted light is collected in the fluorescence-2 (FL2) channel.

PE-Texas Red™ is a tandem conjugate system which combines PE and Texas Red™ and has an absorption maximum of approximately 564 nm. When excited by 488-nm light, the excited fluorochrome (PE) is able to transfer its fluorescent energy to the Texas Red™ molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 615 nm. Special care must be taken when using PE-Texas Red™ conjugates in conjunction with PE as there is considerable spectral overlap in the emission profiles of both fluorochromes.

Peridinin chlorophyll protein (PerCP) is a component of the photosynthetic apparatus found in the dinoflagellate,Glenodinium . PerCP is a protein complex with a molecular weight of approximately 35 kDa. When excited by light at 488 nm from an argon-ion laser, PerCP has a excitation maximum around 490 nm, with an emission spectrum which peaks at 675 nm. The emitted light is collected in the fluorescence-3 (FL3) channel. Due to its photobleaching characteristics, PerCP conjugates are not recommended for use on stream-in-air flow cytometers.

PerCP-Cy5.5 is a tandem conjugate system than combines PerCP with a cyanine dye (Cy5.5™) and has an absorption maximum of approximately 490 nm. When excited by 488-nm light, the excited fluorochrome (PerCP) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 694 nm. Using a 650 nm longpass filter will give optimum detection for this fluorochrome. The emitted light is collected in the fluorescence-3 (FL3) channel. PerCP-Cy5.5 is recommended for use with stream-in-air flow cytometers.

APC-Cy7 is a tandem conjugate system that combines APC and a cyanine dye (Cy7™) and has an absorption maximum of approximately 650 nm. When excited by light from a dye or HeNe laser, the excited fluorochrome (APC) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 767 nm. It is recommended that a 750-nm longpass filter be used along with a red-sensitive detector such as the Hammatsu R3896 PMT for this fluorochrome. Special filters are required when using APC-Cy7 in conjunction with APC. It is recommended that special precautions be taken with PharRed conjugates, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light.

Texas Red™ is a sulfonyl chloride derivative of sulforhodamine 101 with a molecular weight of 625 daltons. BD Biosciences Pharmingen offers Texas Red™ , conjugated to avidin, as a useful second step for multi-color analysis. Because it emits in the long wavelengths of the deep red region (Fig 2), Texas Red™ has little spectral overlap with FITC. When performing multi-color analysis involving both Texas Red™ and PE, BD Biosciences Pharmingen recommends excitation of Texas Red™ using a dual-laser flow cytometer equipped with a tunable dye laser to avoid "leaking" into the PE detector. If a krypton laser, emitting light at 568 nm, is used, the laser light will "leak" into the PE channel. Texas Red™ can be used in conjunction with APC for multi-color analysis when both dyes are excited in the 595-605 nm range with a dye laser. Texas Red™ has an absorption maximum of 596 nm (Fig. 2). Its emission maximum, when excited by 595-600-nm laser light, is 615 nm (Fig. 2). Using a 620 ± 10-nm bandpass filter will give optimum detection for this fluorochrome (Fig. 1).

Comparative staining using a monoclonal antibody (RA3-6B2; anti-B220; Cat. No. 557390/553084**) conjugated to different fluorochromes and analyzed on either BD FACSVantage™ (upper panels) or BD FACSCalibur™ (lower panels). The numbers indicate the ratio of the median fluorescence intensity of positive cells to the negative cells (signal to noise ratio). These plots demonstrate how choices in A) fluorochrome-conjugates or B) instrumentation can affect the fluorescence intensity observed for a given population.

1. The differences observed between individual fluorochrome-conjugates can be affected by the mAb conjugated. Thus while in the example above the PE-conjugate is brighter than the PE-Cy5, when analyzed on the BD FACSCalibur™ , for many mAbs the PE-Cy5-conjugate results in a brighter stain. Contact BD Biosciences Pharmingen Technical Services for more information on specific reagents.
2. Similarly different flow cytometers utilize different lasers and different fluorescence filter sets which can result in differences in signal to noise ratios when using the same reagent. Note that PE reagents tend to be brighter when used on a BD FACSCalibur™ while APC reagents are brighter on a BD FACSVantage™. Note changes of the signal to noise ratio depending on fluorchrome and instrument used.

Figure 1.

"Top schematic." A single laser flow cytometer with five parameters of detection. Two detectors detect the light scatter, and three photo-multiplier tubes (PMTs) detect the fluorescent signals. The bandpass filters are set up for optimal detection with BD Biosciences' fluorochromes: FITC, PE, PE-Cy5 and PerCP.

"Bottom schematic." A dual laser flow cytometer with six parameters of detection. Two detectors detect the light scatter, and four PMTs detect the fluorescent signals. The bandpass filters are set up for optimal detection with fluorochromes FITC, PE, APC, and Texas Red ™ . The second (orange) laser light is emitted from a tunable dye head using rhodamine 6 G as the fluorescent dye for excitation. Forward light scatter (FSC), side scatter (SSC), FITC, and PE signals are all produced by the primary 488-nm argon-ion laser. APC and Texas Red™ signals are produced by the second laser (dye head with a 488-nm argon-ion laser).

Figure 2. Absorption spectra of Fluorochromes. Individual fluorochrome excitation spectra are found in gray and the corresponding emission spectra in black. Typical band pass filters are given for each fluorochrome as used on a FACSVantage™ except for PE-Cy5 and PerCP which are shown for FACSCalibur™ configurations.

TABLE 1. Comparison of individual fluorochromes with single and dual laser flow cytometry.

^*PerCP is highly sensitive to photobleaching and must be used with laser power <150 mW

⁺⁺Can only be used with a dye laser

^#Not recommended (dull)

^$PE-Cy5 and APC cannot be simultaneously used on instruments lacking cross-beam compensation.

References:

Loken, M.R., 1990. Immunofluorescence Techniques in Flow Cytometry and Sorting, 2nd Ed., Wiley. pp 341-353.

Parks, D., L. Herzenberg, and L. Herzenberg. 1989. Flow cytometry and fluorescence-activated cell sorting. Fundamental Immunology, Second Edition. William Paul, Ed.Raven Press, Ltd, New York.

Zola, H. 1995. Detection of cytokine receptors by flow cytometry. In Current Protocols in Immunology (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, eds.) John Wiley and Sons, New York. Unit 6.21.

Immunofluorescence and cell sorting. In Current Protocols in Immunology. (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, eds) John Wiley and Sons, New York. Unit 5.1 - 5.6.5. Shapiro, H.M. 1988. Practical Flow Cytometry, 2nd Ed. Wiley-Liss, New York.

Doucet M, Soussi N, Crain-Denoyelle AM, Gendron MC, Sanchez P. Immunogenetics. 2001. R-phycoerythrin-cyanine 5 tandem discerns CD72 polymorphism. May-Jun; 53(4):307-14.

A valuable forum for discussion of many aspects of flow cytometry is "The Cytometry Electronic Mailing List." To subscribe, contact: cyto-request@flowcyt.cyto.purdue.edu.