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.

The flow cytometer utilizes a variety of lasers, each of which produces monochromatic light.

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). Cells can be separated in to different populations on the basis of light scatter, however flow cytometry is particularly useful to identify specific proteins expressed by the cells or to separate and sort the cells based on protein expression. To achieve this, fluorescently-labeled antibodies are employed to label specific proteins that are expressed by the cells in the sample.


The flow cytometer utilizes a variety of lasers, each of which produces monochromatic light. The laser wavelengths range from ultraviolet to far red.

Laser wavelengths ranging from ultraviolet to far red.

Following absorption of the laser light as the stained cell passes through the beam, the electrons of each fluorochrome become excited; they then produce a transient light emission as they return to the ground state. The emitted light has a longer wavelength than that which was used for excitation, and is detected with specialized sensors.

When selecting suitable fluorochromes for use in a flow cytometry experiment, a number of factors should be taken in to consideration:

  • The available lasers and fluorescence detectors of the flow cytometer
  • The absorption and emission spectra of each fluorochrome
  • The extinction coefficient of each fluorochrome – this defines how well the fluorochrome absorbs light at a particular wavelength; fluorochromes with higher extinction coefficients are generally brighter
  • The Stokes shift of each fluorochrome – this is the difference between the maximum absorbance and emission wavelengths of a fluorochrome; a greater Stokes shift is indicative of less overlap between the two wavelengths

The Stokes shift of each fluorochrome - this is the difference between the maximum absorbance and emission wavelengths of a fluorochrome


Multi-color flow cytometry relies on the use of a combination of fluorescently-labeled antibodies to detect different protein targets within the same sample. When using multiple fluorochromes simultaneously it is important to consider the characteristics of each fluorochrome, and their compatibility with one another, as well as the nature of the flow cytometer itself.

As shown in the figure above(left), BPE and Cy3 fluorescent proteins show very similar peaks of absorbance and emission, making detection of the two dyes practically indistinguishable. Conversely, APC and AMCA fluorescent proteins (right) absorb and emit at the different wavelengths, making the combination of these two proteins ideal for multi-color flow cytometry detection.

A limitation of multi-color flow cytometry

Despite the technological advances in instrument capability, the availability of conjugates for multi-color flow cytometry has not followed suit and it is not unusual to find that the specific fluorochrome conjugate which you need is not available.

The solution?

We have developed a technology which allows direct conjugation of your antibody, protein or peptide to a fluorescent label in under 20 minutes for use in multi-color flow cytometry!

Our Lightning-Link® antibody labeling kits allow direct conjugation of primary antibodies, proteins or peptides with only 30 seconds hands-on time.

We have over 35 fluorescent dyes in our expanding range, therefore if the antibody conjugate you require isn’t available on the market, you can now prepare your own conjugate in less than 20 minutes, with a 100% yield from the unconjugated antibody! The simplicity and convenience of Lightning-Link® antibody labeling kits therefore provides additional ease and flexibility in panel design for multi-colour flow cytometry.

To prepare the conjugate, simply pipette the antibody into a vial of lyophilized mixture containing the label of interest, incubate for 15 minutes (when using our Lightning-Link® rapid range, for standard Lightning-Link® kits incubate for 3 hours) and then stop the reaction. With no desalting or dialysis steps, your conjugate is then ready to use! Furthermore there is no need for any modification of your antibody in advance, unlike many other systems. V


The power of Lightning-Link® for multi-color flow cytometry is clearly demonstrated in a publication by Robinson et al., who used eight different conjugates to undertake detailed analysis of oligodendroglial cell populations in murine brains (Robinson, PA et al (2014) PLOS One 9:1-14).

The strategy is based on the conjugation of each antibody to a unique fluorochrome for rapid analysis of multiple proteins. As shown in the image below (Robinson et al., Fig. 4-D), cells were isolated, stained with oligodendroglial antibodies, and sorted by FACS into three populations: A2B5+PDGFRα+ early oligodendrocyte progenitor cells (OPCs) , NG2+O4+ intermediate OPCs, and GALC+MOG+ mature oligodendrocytes.

Representatives sorting flow plots

Representative sorting flow plots. Rapid analysis of multiple proteins.

For more information on multi-color flow cytometry and considerations of use – take a look at our beginner’s guide to flow cytometry

View the full range of Lightning-Link® fluorescent dyes including high sensitivity DyLight®, Atto® and Cy® dyes and try our new Spectra Viewer tool.

More questions?

Please feel free to contact us. We have an experienced team of scientists and a technical support team ready to answer all your questions regarding our range of products and their applications