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.

Fluorophore Brightness

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. Learn more about BioLegend’s Tandem Dyes.

View our complete Brightness Index


 

Our technical service team is available to help with any questions you may have on multicolor flow cytometry.

Technical Service
Phone Toll-Free (US & Canada)1-877-273-3103
Phone (International): 1-858-768-5801 
Email: Click Here

For a complete listing of the trademarks and patents referenced on this page, click here.

PE/Fire™ 780 is an equivalent fluor to PE/Cy7. It is restricted to ASR use only and is only available to US customers.

Your Instrument

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, use our Instrument Guide/Fluorophore Selector Tool linked below, check your cytometer manual, or check with your local flow core facility. Click on the image below to use the Instrument Guide/Fluorophore Selector Tool.

 


Our technical service team is available to help with any questions you may have on multicolor flow cytometry.

Technical Service
Phone Toll-Free (US & Canada)1-877-273-3103
Phone (International): 1-858-768-5801 
Email: Click Here

For a complete listing of the trademarks and patents referenced on this page, click here.

PE/Fire™ 780 is an equivalent fluor to PE/Cy7. It is restricted to ASR use only and is only available to US customers.

Webinars


Our technical service team is available to help with any questions you may have on multicolor flow cytometry.

Technical Service
Phone Toll-Free (US & Canada)1-877-273-3103
Phone (International): 1-858-768-5801 
Email: Click Here

For a complete listing of the trademarks and patents referenced on this page, click here.

PE/Fire™ 780 is an equivalent fluor to PE/Cy7. It is restricted to ASR use only and is only available to US customers.
 

Expression of Common Markers

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.

View Expression of Common Surface Molecules on Blood Cells


Our technical service team is available to help with any questions you may have on multicolor flow cytometry.

Technical Service
Phone Toll-Free (US & Canada)1-877-273-3103
Phone (International): 1-858-768-5801 
Email: Click Here

For a complete listing of the trademarks and patents referenced on this page, click here.

PE/Fire™ 780 is an equivalent fluor to PE/Cy7. It is restricted to ASR use only and is only available to US customers.

The Rules

There are a few basic rules to follow when setting up a multicolor panel. Follow these rules to organize and optimize your results.

  1. 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.
  2. 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.
  3. 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.
  4. Avoid acidic buffer conditions with FITC labeled samples because FITC is sensitive to low pH.
  5. 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.
  6. Avoid incubating cells in fixative for extended periods of time, as this may affect fluorescence, particularly of tandems dyes.

Our technical service team is available to help with any questions you may have on multicolor flow cytometry.

Technical Service
Phone Toll-Free (US & Canada)1-877-273-3103
Phone (International): 1-858-768-5801 
Email: Click Here

For a complete listing of the trademarks and patents referenced on this page, click here.

PE/Fire™ 780 is an equivalent fluor to PE/Cy7. It is restricted to ASR use only and is only available to US customers.

For more useful tools and information, see BioLegend’s complete set of webtools, as well as other external resources provided below.

Fluorescence Spectra Analyzer

 

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.

 


 Multicolor Panel Selector

 

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.

 


Purdue Archive

 

Purdue University Cytometry Laboratories’ Email Archive Search through the email archive for questions and discussions, or ask your own question to the flow cytometry community.


Purdue Cytometry Archives


 

United States Organizations

International Organizations


Literature

 

Books

  1. Darzynkiewicz Z,Roederer M,Tanke H,eds. Cytometry. 4th ed. Boston: Elesevier Academic Press;2004. ISBN:0125641702.
  2. Darzynkiewicz Z.Flow Cytometry. 2nd ed. San Diego,Ca: Academic Press;1994. ISBN:0125641427.
  3. Darzynkiewicz Z,Chrissman HA,Robinson JP,eds. Methods in Cell Biology:Cytometry. 3rd ed. San Diego,CA: Academic Press;200;63 (PT.A).
  4. Givan AL.Flow Cytometry:First Principles. New York,2nd ed. NY:Wiley-Liss;2001. ISBN0471382248.
  5. Grogan WM, Collins JM. Guide to Flow Cytometric Methods. New York,NY:Marcel Dekker;19890. ISBN 0824783301.
  6. 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.
  7. Nunez R. Flow cytometry for research scientists:principles and applications. Norwich,UK; Horizon Scientific Press;2001. ISBN 1898486263.
  8. Riley RS. Clinical Applications for Flow Cytometry.New York,NY:Igaku-Shoin;1993. ISBN 0896402002.
  9. Shapiro H.Practical Flow Cytometry. 4th ed. New York,NY: Alan R. Liss; 2003. ISBN 0471411256.
  10. Watson JV. Introduction to Flow Cytometry. New York,NY:Cambridge University Press;1991. ISBN 0521380618.


Periodicals

  1. Cytometry Part B: Clinical Cytometry
  2. Current Protocols in Cytometry
  3. Current Protocols in Immunology
  4. Cytometry Part A
  5. Journal of Immunoassay and Immunochemistry

Our technical service team is available to help with any questions you may have on multicolor flow cytometry.

Technical Service
Phone Toll-Free (US & Canada)1-877-273-3103
Phone (International): 1-858-768-5801 
Email: Click Here

For a complete listing of the trademarks and patents referenced on this page, click here.

PE/Fire™ 780 is an equivalent fluor to PE/Cy7. It is restricted to ASR use only and is only available to US customers.

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