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Flow Cytometry

Flow cytometry is a technique which is used to analyze the characteristics of cells as they flow singly past one or more beams of focused light, provided by lasers. When the cells pass through the laser beam they scatter the light, which is detected as forward scatter (correlating to cell size) and side scatter (correlating to granularity). Light scatter can be used to separate cells into different populations. To learn more about flow cytometry, why not download our free guide, or watch our webinar?

Flow cytometric separation of cells in to different populations based on light scatter.

Figure 1. Flow cytometric separation of cells into different populations based on light scatter.

In addition to light scatter, cellular characteristics can be further defined by staining with antibodies against specific proteins. Antibody binding is typically detected via a fluorometric readout. For more information regarding the selection of suitable fluorochromes for a flow cytometry experiment, why not read our multi-color flow cytometry application note?

Indirect versus direct detection

The detection method used within a flow cytometry experiment can be either indirect or direct. Indirect detection relies on the use of a labeled secondary antibody, however these reagents can be a source of background signal, resulting in decreased assay sensitivity. During direct detection, a labeled primary antibody is used, and this method of detection provides several clear advantages:

  • Non-specific binding is avoided since secondary antibodies are not used
  • Multiplexing is possible with antibodies from the same species
  • Faster since there is no secondary antibody incubation step and therefore fewer wash steps
  • Data quality is improved through assay simplification

 

Indirect versus direct staining for flow cytometry.

Figure 2. Indirect versus direct staining for flow cytometry. In this example, antibodies have been used to distinguish between two different cell types, shown in pink and green. The image on the left illustrates indirect staining – labeled secondary antibodies, shown in blue, have been used to detect binding of the specific primary antibodies, shown in black and grey. Cross-reactivity of one secondary antibody (labeled with a red fluorochrome) with the other secondary antibody (labeled with an orange fluorochrome) can produce misleading results. The image on the right illustrates direct staining – the use of directly labeled primary antibodies eliminates the need for secondaries, avoiding non-specific staining.

Lightning-Link® for direct antibody labeling

Lightning-Link® from Expedeon is an innovative technology that enables direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups. The product range includes kits for labeling antibodies with fluorescent dyes, fluorescent proteins, enzymes, biotin or streptavidin, allowing quick and easy production of antibody conjugates which are suitable for use in a wide variety of applications, including flow cytometry.

The Lightning-Link® range includes almost 40 different fluorescent labels, around 20% of which are tandem dyes. The inclusion of tandems in a flow cytometry experiment is an effortless way of increasing the quantity of readouts, especially if the number of excitation lasers is a limiting factor. The maximal absorption and emission wavelengths, Stokes shift data and extinction co-efficients for all our Lightning-Link® fluorophores can be found in our fluorescence table.

 

 

To produce the conjugate, the antibody is simply pipetted into a vial of lyophilized Lightning-Link® mixture containing the label of interest, incubated, and is then ready for use.

The Lightning-Link® conjugation process.

 

The use of directly labeled primary antibodies demonstrates a similar level of staining to that which is produced when labeled secondaries are used, yet provides all the advantages discussed above.

 

Flow cytometry data comparing indirect and direct cellular staining.

Figure 4. Flow cytometry data comparing indirect and direct cellular staining. A CD45RC antibody was purchased in unconjugated form from a commercial source, and was conjugated using a Lightning-Link® R-PE antibody labeling kit according to the kit protocol. Indirect staining using an R-PE-labeled secondary antibody (left) was compared with direct staining using the R-PE-conjugated CD45RC antibody (right). Similar levels of staining were obtained in both scenarios.


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Multi-color flow cytometry

Directly label primary antibodies for multi-color flow cytometry Flow cytometry is an extremely powerful technique that is used to analyze the characteristics of cells as they flow singly past one or more beams of focused light, provided by lasers. When the cells pass through the laser beam they scatter the light, which is detected as…

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A-Z Guide to Fluorochromes

August 23rd, 2017

        Download a Free Copy Lightning-Link® fluorescent antibody and protein labeling kits allow you to covalently label any antibody in under 20 minutes with only 30 seconds hands-on time. We currently have over 35 label in our range of fluorescent dyes. This guide provides a general overview of each fluorochrome in our…

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Absorption-Emission Table

August 23rd, 2017

Lightning-Link® is an innovative technology that enables direct labeling of antibodies, proteins, peptides or other biomolecules with only 30 seconds hands-on time.

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Simplifying Antibody Conjugation Process

August 23rd, 2017

Antibodies are widely employed in the quantification of antigens in complex biological samples. Using techniques such as Western blotting, ELISA, and immunohistochemistry researchers are able to measure a single antigen, or perhaps a limited number of antigens, in each sample. In the post-genomics era, advances in multiplex immunoassay technologies now allow scores or even hundreds…

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Antibody Purification Guide

August 23rd, 2017

        Download your copy Expedeon specializes in easy to use bioconjugation kits which enable the direct labeling of antibodies or proteins with enzymes, fluorescent labels, biotin, streptavidin, gold nanoparticles, latex beads or oligonucleotides. Unfortunately, many antibodies are provided in buffers which contain additives that are incompatible with labeling technologies, making puri cation…

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Guide to labeling your primary antibody

August 23rd, 2017

        Download a free copy! Antibodies are used to detect and quantify antigens in techniques such as flow cytometry, ELISA, western blotting, immunohistochemistry and lateral flow. The antibody that binds to the antigen is called a ‘primary antibody’ and it confers specificity to the assay. Most types of immunoassays also incorporate a…

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A Beginner’s Guide to Flow Cytometry

August 17th, 2017

Flow Cytometry is a widely used method for cell analysis which, for the novice, can appear daunting due to the complexity of the experimental approach and data analysis.

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The advantages of tandem dyes

August 9th, 2017

Blog

A significant number of laboratory applications rely on the use of fluorescently-labeled antibodies.

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Antibody labeling protocol video

May 7th, 2018

Applications, Videos

Stop using secondary antibodies in immunoassays! Save time and materials by labeling your primary antibodies in under 20 min. For use in ELISA, western blotting, IHC, flow cytometry and many other applications. Scientists love our Lightning-Link® kits because of the remarkable simplicity of the technology. Now they can open a…

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A Beginner’s Guide to Flow Cytometry

September 24th, 2012

Applications, Videos, Webinars

**This webinar was produced by Innova Biosciences which is now fully integrated with their sister company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   This on-demand webinar brings together a unique combination of content from two speakers with more than 50 years of experience in flow…

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Flow cytometry – Sample preparation & experimental design

February 17th, 2014

Applications, Webinars

**This webinar was produced by Innova Biosciences which is now fully integrated with their company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   This advanced class discusses flow cytometry  sample preparation and the process of experimental design to ensure a successful flow cytometry experiment. Webinar topics…

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Methods of antibody conjugation – which one is best for you?

January 20th, 2014

Webinars

**This webinar was produced by Innova Biosciences which is now fully integrated with their company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   Dr Nick Gee, the CEO and CSO of Expedeon, explains the benefits of using directly conjugated antibodies and the various approaches and techniques…

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How to Overcome all your Problems with Secondary Antibodies

December 6th, 2012

Applications, Webinars

**This webinar was produced by Innova Biosciences which is now fully integrated with our sister company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   This webinar discusses how to overcome problems associated with using secondary antibodies in your immunoassay, including: Secondary antibodies require a series of…

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A Beginner’s Guide to Flow Cytometry

September 24th, 2012

Applications, Videos, Webinars

**This webinar was produced by Innova Biosciences which is now fully integrated with their sister company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   This on-demand webinar brings together a unique combination of content from two speakers with more than 50 years of experience in flow…

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