Multicolor Flow Cytometry
Spectrum Guide
 | When making decisions about which fluorochromes to use in your experiments, you'll want to know their relative emission spectra. Simply mouse over the spectrum on the left side to view histograms that represent the absorption and emission spectra for each BD™ fluorochrome. | ||
  |  | BD Horizon™ V450 (Em-Max 448 nm) is a new coumarin dye excited by the violet laser that exhibits spectral properties similar to Pacific Blue™. The BD Horizon V450 dye has an excellent quantum yield of nearly 1 (0.99), and conjugates are typically as bright or brighter than comparable reagents conjugated to Pacific Blue™. | |
 | | Pacific Blue™ (Em-Max 452 nm), a dye excited by the violet laser, is based on the 6,8-difluoro-7-hydroxycoumarin fluorophore, and is strongly fluorescent, even at neutral pH. | |
 | | AmCyan (Em-Max 491 nm) is a 108-kDa protein derived from Anemonia majano. With an excitation peak of 458 nm and an emission peak of 491 nm, it can be used in combination with BD Horizon V450 or Pacific Blue™ on violet laser–equipped flow cytometers. | |
| | BD Horizon™ V500 (Em-Max 500 nm) is a novel organic dye excited by the violet laser with maximum excitation at 415 nm and maximum emission at 500 nm. This dye offers improved brightness over Pacific Orange™ and reduced spillover into the FITC channel when compared to AmCyan. | |
| | Alexa Fluor® 488 (Em-Max 519 nm) conjugates are highly photostable and remain fluorescent over a broad pH range. Due to its extraordinary photostability, this fluorochrome is also highly suitable for fluorescence microscopy. | |
 | | FITC (Em-Max 520 nm), fluorescein isothiocyanate, is a fluorochrome with a molecular weight of 389 Da. 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 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. FITC is sensitive to pH changes and photobleaching. | |
 | | R-PE (Em-Max 578 nm), R-phycoerythrin 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 R-phycoerythrin an ideal pigment for flow cytometry applications. | |
 | | PE-Texas Red® (Em-Max 615 nm) is a tandem conjugate system that combines R-PE and Texas Red®. Special care must be taken when using PE-Texas Red® conjugates in conjunction with R-PE since there is considerable spectral overlap in the emission profiles of both fluorochromes. | |
 | | Texas Red® (Em-Max 620 nm) is a sulfonyl chloride derivative of sulforhodamine 101 with a molecular weight of 625 kDa. Texas Red® conjugated to avidin is a useful second step for multicolor analysis. When performing multicolor analysis involving both Texas Red® and R-PE, use of a dual-laser flow cytometer equipped with a tunable dye laser to avoid “leaking” into the PE detector is recommended. If a krypton laser emitting light at 568 nm is used, the laser light will leak into the R-PE channel. Texas Red® can be used in conjunction with APC for multicolor analysis when both dyes are excited in the 595–605 nm range with a dye laser. | |
| | APC (Em-Max 660 nm), allophycocyanin, is an accessory photosynthetic pigment found in blue-green algae. Its molecular weight is approximately 105 kDa. APC has six phycocyanobilin chromophores per molecule, which are similar in structure to phycoerythrobilin, the chromophore in R-PE. | |
| | PE-Cy™5 (Em-Max 667 nm), formerly BD Cy-Chrome, is a tandem conjugate system that combines R-phycoerythrin (a 240-kDa protein) and a cyanine dye (MW 1.5 kDa). The efficiency of the light energy transfer between the two fluorochromes allows less than 5% of the absorbed light to be lost as fluorescence at 575 nm by R-PE. As with our R-PE conjugates, an average of one PE-Cy5 molecule is coupled per antibody or protein. Because of its broad absorption range, PE-Cy5 is not recommended for use with dual-laser flow cytometers where excitation by both lasers is possible. PE-Cy5 is not recommended for routine fluorescence microscopy because it is subject to photobleaching. | |
| | Alexa Fluor® 647 (Em-Max 668 nm) conjugates are highly photostable and remain fluorescent over a broad pH range. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. | |
| | PerCP (Em-Max 678 nm) 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. Due to its photobleaching characteristics, PerCP conjugates are not recommended for use on stream-in-air flow cytometers. | |
 | | PerCP-Cy™5.5 (Em-Max 695 nm) is a tandem conjugate system that combines PerCP with a cyanine dye (Cy5.5). PerCP-Cy5.5 can be used with stream-in-air flow cytometers. PerCP-Cy5.5 is not subject to photobeaching like PerCP, and it causes less Fc receptor-mediated nonspecific staining than PE-Cy5. Additionally, the PerCP-Cy5.5 tandem conjugate is not as susceptible to fixative or light instability compared to APC-Cy7 and PE-Cy7. | |
 | | Alexa Fluor® 700 (Em-Max 723 nm) is a far-red dye that has a peak absorption maximum of 696–700 nm, can be excited with a HeNe or red diode laser, and has a peak emmission at 719–720 nm. This unique emission spectrum enables multicolor analysis in conjunction with APC or Alexa Fluor® 647, and APC-Cy7 reagents. | |
| | APC-Cy™7 (Em-Max 785 nm) is a tandem conjugate system that combines APC and a cyanine dye (Cy7). We recommend that special precautions be taken with APC-Cy7 conjugates, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light. Caution: Some APC-Cy7 conjugates show changes in their emission spectra with prolonged exposure to formaldehyde. We recommend that you analyze fixed samples within four hours. | |
| | APC-H7 (Em-Max 785 nm) is a new APC-cyanine tandem dye and an analog of APC-Cy7 with similar spectral properties. APC-H7 has been engineered for greater stability in light and paraformaldehyde fixatives and less spillover in the APC channel. Conjugates of APC-H7 are typically 25% less bright than equivalent APC-Cy7 conjugates. | |
 | | PE-Cy™7 (Em-Max 785 nm) is a tandem conjugate system that combines PE and a cyanine dye (Cy7). PE-Cy7 conjugated reagents are as bright as PE conjugates. PE-Cy7 can be used simultaneously with FITC, PE, PE-Cy5, PerCP, PerCP-Cy5.5, PE-Texas Red®, APC, and APC-Cy7 with minimal crossbeam compensation. PE-Cy7 is particularly sensitive to photo-induced degradation. Extreme caution must be taken to avoid light exposure. | |
BD Horizon™ V450 (Em-Max 448 nm) is a new coumarin dye excited by the violet laser that exhibits spectral properties similar to Pacific Blue™. The BD Horizon V450 dye has an excellent quantum yield of nearly 1 (0.99), and conjugates are typically as bright or brighter than comparable reagents conjugated to Pacific Blue™.
Pacific Blue™ (Em-Max 452 nm), a dye excited by the violet laser, is based on the 6,8-difluoro 7-hydroxycoumarin fluorophore, and is strongly fluorescent, even at neutral pH.
AmCyan (Em-Max 491 nm) is a 108-kDa protein derived from Anemonia majano. With an excitation peak of 458 nm and an emission peak of 491 nm, it can be used in combination with BD Horizon V450 or Pacific Blue™ on violet laser–equipped flow cytometers.
BD Horizon™ V500 (Em-Max 500 nm) is a novel organic dye excited by the violet laser with maximum excitation at 415 nm and maximum emission at 500 nm. This dye offers improved brightness over Pacific Orange™ and reduced spillover into the FITC channel when compared to AmCyan.
Alexa Fluor® 488 (Em-Max 519 nm) conjugates are highly photostable and remain fluorescent over a broad pH range. Due to its extraordinary photostability, this fluorochrome is also highly suitable for fluorescence microscopy.
FITC (Em-Max 520 nm), fluorescein isothiocyanate, is a fluorochrome with a molecular weight of 389 kDa. 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 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. FITC is sensitive to pH changes and photobleaching.
R-PE (Em-Max 578 nm), R-phycoerythrin 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 R-phycoerythrin an ideal pigment for flow cytometry applications.
PE-Texas Red® (Em-Max 615 nm) is a tandem conjugate system that combines R-PE and Texas Red®. Special care must be taken when using PE-Texas Red® conjugates in conjunction with R-PE since there is considerable spectral overlap in the emission profiles of both fluorochromes.
Texas Red (Em-Max 620 nm) is a sulfonyl chloride derivative of sulforhodamine 101 with a molecular weight of 625 kDa. Texas Red® conjugated to avidin is a useful second step for multicolor analysis. When performing multicolor analysis involving both Texas Red® and R-PE, use of a dual-laser flow cytometer equipped with a tunable dye laser to avoid “leaking” into the PE detector is recommended. If a krypton laser emitting light at 568 nm is used, the laser light will leak into the R-PE channel. Texas Red® can be used in conjunction with APC for multicolor analysis when both dyes are excited in the 595–605 nm range with a dye laser.
APC (Em-Max 660 nm), allophycocyanin, is an accessory photosynthetic pigment found in blue-green algae. Its molecular weight is approximately 105 kDa. APC has six phycocyanobilin chromophores per molecule, which are similar in structure to phycoerythrobilin, the chromophore in R-PE.
PE-Cy™5 (Em-Max 667 nm), formerly BD Cy-Chrome, is a tandem conjugate system that combines R-phycoerythrin (a 240-kDa protein) and a cyanine dye (MW 1.5 kDa). The efficiency of the light energy transfer between the two fluorochromes allows less than 5% of the absorbed light to be lost as fluorescence at 575 nm by R-PE. As with our R-PE conjugates, an average of one PE-Cy5 molecule is coupled per antibody or protein. Because of its broad absorption range, PE-Cy5 is not recommended for use with dual-laser flow cytometers where excitation by both lasers is possible. PE-Cy5 is not recommended for routine fluorescence microscopy because it is subject to photobleaching.
Alexa Fluor® 647 (Em-Max 668 nm) conjugates are highly photostable and remain fluorescent over a broad pH range. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy.
PerCP (Em-Max 678 nm) 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. Due to its photobleaching characteristics, PerCP conjugates are not recommended for use on stream-in-air flow cytometers.
PerCP-Cy™5.5 (Em-Max 695 nm) is a tandem conjugate system that combines PerCP with a cyanine dye (Cy5.5). PerCP-Cy5.5 can be used with stream-in-air flow cytometers. PerCP-Cy5.5 is not subject to photobeaching like PerCP, and it causes less Fc receptor-mediated non specific staining than PE-Cy5. Additionally, the PerCP-Cy5.5 tandem conjugate is not as susceptible to fixative or light instability compared to APC-Cy7 and PE-Cy7.
Alexa Fluor® 700 (Em-Max 723 nm) is a far-red dye that has a peak absorption maximum of 696–700 nm, can be excited with a HeNe or red diode laser, and has a peak emmission at 719–720 nm. This unique emission spectrum enables multicolor analysis in conjunction with APC or Alexa Fluor® 647, and APC-Cy7 reagents.
APC-Cy™7 (Em-Max 785 nm) is a tandem conjugate system that combines APC and a cyanine dye (Cy7). We recommend that special precautions be taken with APC-Cy7 conjugates, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light. Caution: Some APC-Cy7 conjugates show changes in their emission spectra with prolonged exposure to formaldehyde. We recommend that you analyze fixed samples within four hours.
APC-H7 (Em-Max 785 nm) is a new APC-cyanine tandem dye and an analog of APC-Cy7 with similar spectral properties. APC-H7 has been engineered for greater stability in light and paraformaldehyde fixatives and less spillover in the APC channel. Conjugates of APC-H7 are typically 25% less bright than equivalent APC-Cy7 conjugates.
PE-Cy™7 (Em-Max 785 nm) is a tandem conjugate system that combines PE and a cyanine dye (Cy7). PE-Cy7 conjugated reagents are as bright as PE conjugates. PE-Cy7 can be used simultaneously with FITC, PE, PE-Cy5, PerCP, PerCP-Cy5.5, PE-Texas Red®, APC, and APC-Cy7 with minimal crossbeam compensation. PE-Cy7 is particularly sensitive to photo-induced degradation. Extreme caution must be taken to avoid light exposure.
Cy™ is a trademark of Amersham Biosciences Corp.
Alexa Fluor®, Texas Red®, Pacific Blue™, and Pacific Orange™ are registered trademarks or trademarks of Molecular Probes, Inc.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
