With wide varieties of antibodies, fluorophores, and instruments available for multicolor flow cytometry, optimizing your multicolor experiments can be confusing and frustrating. Use our Multicolor Staining Guide to assist you in developing and optimizing your flow cytometry experiments. Master the five aspects of the Staining Guide and you will be well on your way to legendary discovery. When you are ready to find antibodies, go to the Multicolor Panel Selector. |
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| Before undertaking any flow cytometry experiments, it is important to know the capabilities of your instrument. You will need to define exactly which fluorophores you can or cannot use on your available flow cytometers. To find more information, check your cytometer manual, check with your local flow core facility, or view our “Fluorophores for Flow Cytometry” poster below. The poster lists possible fluorophores for use based on default configurations for the listed instruments. Please note that your specific instrument may have unique configurations that are different from the default settings. Click on the image below to view a larger version. |
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| There are a few basic rules to follow when setting up a multicolor panel. Follow these rules to organize and optimize your results. |
- Choose the brightest fluorophores that can be used on your instrument. In our Brightness Index chart, the brightest fluorophores are Brilliant Violet 421™, PE, and APC, so these should considered first.
- Choose the brightest fluorophore for your least expressed protein and the dimmest fluorophore for your most highly expressed protein. To help you, we have compiled a chart to indicate the expression of common surface molecules on blood cells. Generally, molecules like CD45, CD3, CD4, and CD8 are quite highly expressed on their target cell types and can be used quite successfully with dim fluorophores.
- Choose fluorophores with emissions having the least spectral overlap. For example, although Brilliant Violet 421™ and Pacific Blue™ do not have exact the same emission spectra, they did have significant overlap, so you should generally avoid using these together. Use our Spectra Analyzer to view and compare excitation and emission spectra, view laser lines, and add custom bandpass filters.
- Use tandems (PE/Cy5, PE/Cy7, APC/Cy7) with caution, as they are more susceptible to degradation by light exposure or fixation. They are essential to large multicolor panels, so just use them with care to prevent light exposure and use appropriate fixation buffers and protocols.
- Avoid acidic buffer conditions with FITC labeled samples because FITC is sensitive to low pH.
- Avoid exposing stained samples to bright light as most fluorophores are susceptible to photobleaching, causing them to lose fluorescence. This is particularly true for tandems dyes.
- Avoid incubating cells in fixative for extended periods of time, as this may affect fluorescence, particularly of tandems dyes.
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| Knowing the relative expression of antigen markers can help you optimize your fluorophore selections in multicolor flow cytometry. In general, select bright fluorophores for your weakly expressed antigens and select dimmer fluorophores for highly expressed antigens. This strategy provides more manageable compensation for your samples and gives you the most accurate data for weakly expressed antigens. |
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| It is important to understand the properties and capabilities of each fluorophore that you may use for multicolor flow cytometry. One such important property is the relative brightness of the fluorophore when conjugated to antibodies. We provide these value in terms of Relative Brightness Index. Brightness Index values are not absolute and can vary depending on the antibody, the antigen, the instrument and its configuration, the staining protocol, the cell type, and other factors. The Brightness Index is provided as a general guide to help in multicolor panel construction, with 1 being the dimmest and 5 being the brightest. The tables below also provide emission spectra and important comments on the use of each fluorophore. Explore fluorophores by laser excitation with the tabs below. Also use our Fluorescence Spectra Analyzer to compare and analyze excitation and emission data. |
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| Fluorophore |
Brightness Index (1=dim, 5=bright) |
Exmax |
Emmax |
Comments |
| Brilliant Violet 421™ |
5 |
405 nm |
421 nm |
Brilliant Violet 421™ is a novel organic fluorescent polymer capable of extremely bright signal and high photostability. It is an excellent choice for flow cytometry and microscopy. It is not recommended for use with Pacific Blue™. |
| Pacific Blue™ |
1 |
401 nm |
455 nm |
Pacific Blue™ is a hydroxycoumarin-based fluorophore, generally considered to be dimmer than most other fluorophores. It is recommended for highly expressed antigens. Brilliant Violet 421™ is a brighter alternative for weakly expressed antigens. |
| Brilliant Violet 570™ |
3 |
405 nm |
570 nm |
Brilliant Violet 570™ is a novel organic fluorescent polymer. It provides a much brighter alternative to Pacific Orange™ for multicolor flow on the violet laser and is a better alternative to nanocrystals for intracellular flow cytometry. |
| Brilliant Violet 605™ |
5 |
405 nm |
603 nm |
Brilliant Violet 605™ is a novel organic fluorescent polymer. It provides a much brighter alternative to eFluor® 605 for multicolor flow on the violet laser and is a better alternative to nanocrystals for intracellular flow cytometry. |
| Brilliant Violet 650™ |
4 |
405 nm |
646 nm |
Brilliant Violet 650™ is a novel organic fluorescent polymer. It provides a much brighter alternative to eFluor® 650 for multicolor flow on the violet laser and is a better alternative to nanocrystals for intracellular flow cytometry. |
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| Fluorophore |
Brightness Index (1=dim, 5=bright) |
Exmax |
Emmax |
Comments |
| Alexa Fluor® 488 |
3 |
495 nm |
519 nm |
Alexa Fluor® 488 is a fluorescent molecule with exceptional photostability. For flow cytometry it has very similar brightness and emission properties compared to FITC, but is advantageous for microscopy applications. |
| FITC |
3 |
494 nm |
520 nm |
FITC (fluorescein isothiocyanate) is a small synthetic organic fluorophore with moderate brightness, suitable for flow and microscopy applications. It is sensitive to pH changes. It cannot be used with Alexa Fluor® 488. |
| PerCP |
2 |
482 nm |
678 nm |
PerCP is a naturally-derived carotenoid pigment found in photosynthetic dinoflagellates. It is not recommended for high powered lasers >25 mW due to its sensitivity to photobleaching. PerCP has overlapping emission with PE/Cy5, so these should not be used on the same laser. |
| PerCP/Cy5.5 |
3 |
482 nm |
695 nm |
PerCP/Cy5.5 is tandem conjugate of PerCP and the cyanine dye, Cy5.5. Unlike PerCP, it is quite photostable and can be used with high powered lasers. Of the all tandems, it is the most photostable and resistant to degradation by fixation. PerCP and PerCP/Cy5.5 have significant overlap and are not recommended for use together. |
| PE |
5 |
496 nm |
578 nm |
R-Phycoerythrin (PE) is a pigment derived from red algae. It is a large 240 kD protein that contains 23 phycoerthrobilin chromophores per molecule. It is extremely bright for flow cytometry, but poor photostability makes it unsuitable for microscopy. PE and its tandem are also sensitive to denaturation by fixation. Because of its wide excitation range, it can be excited by the 488, 532, and 561 nm lasers. |
| PE/Cy5 |
5 |
496 nm |
667 nm |
PE/Cy5 is a tandem conjugate of PE and the cyanine dye, Cy5. Similar to PE, it is bright for flow cytometry, but is sensitive to photobleaching and not recommended for microscopy. Cy5 has been known to non-specifically bind to Fc receptors. On some instruments, it is not recommended for use with APC, due to their overlapping emissions. |
| PE/Cy7 |
4 |
496 nm |
785 nm |
PE/Cy7 is a tandem conjugate of PE and the cyanine dye, Cy7. Similar to PE, it is bright for flow cytometry, but is sensitive to photobleaching and not recommended for microscopy. This tandem is particularly sensitive to light exposure and prolonged fixation. |
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| Fluorophore |
Brightness Index (1=dim, 5=bright) |
Exmax |
Emmax |
Comments |
| PE |
5 |
496 nm |
578 nm |
R-Phycoerythrin (PE) is a pigment derived from red algae. It is a large 240 kD protein that contains 23 phycoerthrobilin chromophores per molecule. It is extremely bright for flow cytometry, but poor photostability makes it unsuitable for microscopy. PE and its tandem are also sensitive to denaturation by fixation. Because of its wide excitation range, it can be excited by the 488, 532, and 561 nm lasers. |
| PE/Cy5 |
5 |
496 nm |
667 nm |
PE/Cy5 is a tandem conjugate of PE and the cyanine dye, Cy5. Similar to PE, it is bright for flow cytometry, but is sensitive to photobleaching and not recommended for microscopy. Cy5 has been known to non-specifically bind to Fc receptors. On some instruments, it is not recommended for use with APC, due to their overlapping emissions. |
| PE/Cy7 |
4 |
496 nm |
785 nm |
PE/Cy7 is a tandem conjugate of PE and the cyanine dye, Cy7. Similar to PE, it is bright for flow cytometry, but is sensitive to photobleaching and not recommended for microscopy. This tandem is particularly sensitive to light exposure and prolonged fixation. |
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| Fluorophore |
Brightness Index (1=dim, 5=bright) |
Exmax |
Emmax |
Comments |
| APC |
5 |
650 nm |
660 nm |
Allophycocyanin (APC) is a 105 kD molecule derived from blue-green algae that has 6 phycocyanobilin chromophores per molecule. It is bright and particularly useful for flow cytometry but not microscopy. It should not be used together with Alexa Fluor® 647 due to their overlapping emissions. |
| Alexa Fluor® 647 |
4 |
650 nm |
668 nm |
Alexa Fluor® 647 is spectrally equivalent to APC, but has much better photostability, making it ideal for microscopy applications. While generally not as bright as APC, it is also a good choice for flow cytometry. |
| Alexa Fluor® 700 |
2 |
696 nm |
719 nm |
Althought it is a dim molecule, Alexa Fluor® 700, expands the choices for the red laser, fitting nicely between APC and APC/Cy7. |
| APC/Cy7 |
2 |
650 nm |
785 nm |
APC/Cy7 is a tandem of APC and the cyanin dye, Cy7. It is particularly sensitive to light and fixation and should be handled with care to avoid light exposure. |
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| For more useful tools and information, see BioLegend’s complete set of webtools, as well as other external resources provided below. |
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Fluorescence Spectra Analyzer
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BioLegend's Fluorescence Spectra Analyzer is useful for the analysis of excitation and emission spectra of commonly used fluorochromes for flow cytometry. You can also create custom bandpass filters, export images, and bookmark your settings. Unlike other fluorescence spectra tools on the internet, this Analyzer does not use Java, which allows it to work well on the iPhone and iPad, viewable from your standard web browser.
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Multicolor Panel Selector
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BioLegend’s Multicolor Panel Selector is a multifaceted tool designed to help you fnd the right products for your multicolor flow cytometry experiments. Featuring:
- Simple interface for panel construction
- Product previews, including data
- Emission spectra preview and fluorophore notes
- Fluorophore brightness index
- Add panel items directly to your shopping cart
- Isotype controls
The tool also provides tips and tricks to constructing optimized multicolor panels.
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A FREE comprehensive online resource for designing multi-parameter flow cytometry experiments and accessing flow cytometry and cell-based analysis products and knowledge. You can also find resources for analysis software, instruments, flow meetings, positions available, and much more. |
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FREE search engine to find antibody suppliers for thousands of specific antibodies. Plus locate sources for ELISA assay kits, cytokines, enzymes, recombinant proteins, siRNAs, tissues, organs, custom antibody services and more. |
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A FREE online resource for multicolor panel design and a web-based communication tool aimed at facilitating an integrated environment for labs and institutions where panels can be shared, inventory can be managed and questions about reagents and instruments can get answered. |
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Heavy duty stand alone flow cytometry analysis software. |
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Discover CyTOF® Mass Cytometry, an alternative to fluorescence-based flow cytometry. A select number of antibodies from BioLegend have been validated on this platform. Use our Advanced Search and select CyTOF® from the applications drop-down menu to find products. |
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Literature
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Books
- Darzynkiewicz Z,Roederer M,Tanke H,eds. Cytometry. 4th ed. Boston: Elesevier Academic Press;2004. ISBN:0125641702.
- Darzynkiewicz Z.Flow Cytometry. 2nd ed. San Diego,Ca: Academic Press;1994. ISBN:0125641427.
- Darzynkiewicz Z,Chrissman HA,Robinson JP,eds. Methods in Cell Biology:Cytometry. 3rd ed. San Diego,CA: Academic Press;200;63 (PT.A).
- Givan AL.Flow Cytometry:First Principles. New York,2nd ed. NY:Wiley-Liss;2001. ISBN0471382248.
- Grogan WM, Collins JM. Guide to Flow Cytometric Methods. New York,NY:Marcel Dekker;19890. ISBN 0824783301.
- Nguyen DT,Diamond LW, Braylan RC. Flow Cytometry in Hematopathology: A Visual Approach to Data Analysis and Interpretation.Totowa,NJ:Human Press;2002. ISBN 1588292126.
- Nunez R. Flow cytometry for research scientists:principles and applications. Norwich,UK; Horizon Scientific Press;2001. ISBN 1898486263.
- Riley RS. Clinical Applications for Flow Cytometry.New York,NY:Igaku-Shoin;1993. ISBN 0896402002.
- Shapiro H.Practical Flow Cytometry. 4th ed. New York,NY: Alan R. Liss; 2003. ISBN 0471411256.
- Watson JV. Introduction to Flow Cytometry. New York,NY:Cambridge University Press;1991. ISBN 0521380618.
Periodicals
- Clinical Cytometry (An official publication of the Clinical Cytometry Society and the International Society for Analytical Cytology) www3.interscience.wiley.com
- Current Protocols in Cytometry www.currentprotocols.com
- Current Protocols in Immunology www.currentprotocols.com
- Cytometry (The journal of International Society for Analytical Cytology) www3.interscience.wiley.com/journal/33945
- Journal of Immunoassay and Immunochemistry www.informaworld.com
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