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Multicolour analysis


The ability to simultaneously measure multiple parameters on a cell by cell basis is probably the most powerful aspect of analytical flow cytometry.  Once more than two colours are being used, the importance of the relevant controls, dye combinations, electronic compensation and optical set-up becomes much more important, and before embarking on such experiments it is vital that you discuss it with the staff of the Flow Cytometry Core Facility. See new webpage on multi-colour experimental design which discusses the pro's and cons of the issues involved, like when to use isotype controls, fluorescence minus one technology, digital compensation with antibody fluorophores, annexin V, DNA and functional dyes.

It is also important to be aware that all fluorochromes will have a range of fluorescence emission and because of the optical filters used in flow cytometers, there will be overlap between the emission spectra once more than one fluorochrome is being used.  In these cases, there will need to be some form of compensation for this spectral overlap. See Roederer M, Darzynkiewicz Z, Parks DR. [new window]. Guidelines for the presentation of flow cytometric data. Methods Cell Biol. 2004;75:241-56. Also go to Compensation on this website for detailed information on the use of the automated compensation software on instruments at ICMS.

The optical filters found in most flow cytometers can be of several types and knowledge of the ones that are used or that can be used will aid in the design and the feasibility of multicolour experiments.

Long Pass filters allow light above a specified wavelength to pass through.  The usual nomenclature is for example, 600LP, where light above 600nm passes through.

Short Pass filters allow light below a specified wavelength to pass through, e.g. 500SP.

Band Pass filters allow light between two wavelengths to pass through.  The usual designation is for example 530/30bp where the centre of the transmitted light is at 530nm and the band is 30nm .i.e light 15nm either side of the centre is transmitted (515-545nm in this example)

Dichroic Filters or Mirrors are also used and these are specialised long or short pass filters.  A 560LP dichroic for example will allow light above 560nm to be transmitted but will reflect all light below 560nm in a specific direction.

The filters available in the cytometers should be checked before selecting the fluorochromes for the analysis.


DD Ateh, VH Leinster, SR Lambert, A Shah, A Khan, H Walklin, J Johnstone, N Ibrahim, M Kadam, Z Malik, M Girones, G Veldhuis, G Warnes, S Marino, I McNeish, JE Martins. The intracellular uptake of CD95 modified paclitaxel-loaded poly(lactic-co-glycolic acid) microparticles Biomaterials 32 (33), 8538-47, 2011.

G Warnes, JP Biggerstaff, JL Francis. Monocyte B7 and Sialyl Lewis X modulates the efficacy of IL-10 down-regulation of LPS-induced monocyte tissue factor in whole blood. British Journal of Haematology 102,588-596, 1998.

Multicolour analysis
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by Gary Warnes. © Queen Mary, University of London 2007
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