University of Dundee

Multi-colour immunofluorescence

The multi-parametric capabilities of flow cytometry makes it a very powerful tool for identifying and quantifying different cell types within heterogeneous populations. Using the LSR flow cytometer up to twelve different colours and hence twelve different antigens or cell characteristics can be detected in the same sample.

However, once more than two colours are being detected at any one time a number of factors come into play that can complicate the analysis. It is therefore important for inexperienced flow cytometrists to seek advice before embarking on multi-colour analysis. The following are just a few of the factors to be taken into consideration.

The use of antibodies

Probably the most common use of multi-parameter flow cytometry exploits the use of fluorescently labelled antibodies for the detection of specific antigens. This means that it is subject to the same 'rules' and pitfalls as all techniques that use this type of methodology. Some of the main considerations are as follows:

  • Dead cells will non-specifically bind antibodies and grossly affect the results obtained, so is important to include a means of distinguishing cells that are alive from those that are dead.
  • Sample controls need to be included to assess the non-specific binding attributable to each of the antibodies in the assay, including second step as well as first step reagents.
  • Fc receptors on some cells (e.g. macrophages, B-cells) will bind antibodies via their Fc region leading to binding unrelated to the antigen specificity. When assessing samples containing such cells it is important to eliminate this by blocking Fc receptors prior to the addition of any other antibodies.

Selection of dyes

For a fluorophore to stand a chance of being detected by a flow cytometer it must first be efficiently excited by one of the light sources available. For different fluorophores to be used in combination with one another they must each have differing emission wavelengths, and this emitted light needs to be at a wavelength that can be detected by the flow cytometer optics. It may also be important to take into account the relative brightness of the signals from each of the fluorophores.

To give a simple example, FITC is a relatively weak fluorophore compared to phycoerythrin (PE), which is very bright. Therefore, if used in combination with one another it is best to use PE to label a weakly expressed antigen, and FITC to label a highly expressed antigen.

Spectral overlap and fluorescence compensation

Even when fluorophores are selected that have different emission spectra, there will still be some spectral overlap between them. For example, FITC is essentially a green fluorophore but it does overlap into the orange (PE) channel slightly. This will make a sample labelled with just FITC look slightly positive for PE.

This spectral overlap can be eliminated during flow cytometric analysis using a process known as 'compensation'. Accurate compensation becomes very important when performing multicolour experiments, particularly when only slight differences in antigen expression are present.

Multicolour Immunoflourescence data

The true power of flow cytometry as a technique becomes apparent when performing multi-colour immunophenotyping. The figure below shows a relatively simple example of five colour analysis looking at the CD62L expression on T cell sub-sets within the heterogenous population of cells derived from murine spleen. By a careful process of population selection by 'gating' the CD62L expression on CD4 and CD8 positive cell sub-sets can be accurately determined.