Three Colour Immunofluorescence on a FACS II Without a Dye Laser
Anne Wilson, Frank Battye, Mark Cozens, Justin Beall and Roland Scollay
The Walter & Eliza Hall Institute
In order to completely define the surface phenotype of some cell populations, it becomes necessary to use more than the standard two-colour immunofluoresence analysis. In combination with Fluorescein and Phycoerythrin, some groups are now using Texas Red as the third fluorochrome in three colour surface staining experiments. Its disadvantage is that it requires dye laser excitation. We now report three colour surface immunofluorescence in the absence of a Dye Laser, but with a FACS II fitted with dual Argon Ion Lasers, one emitting visible light at 488nm, the other laser in the ultra-violet at 350nm.
We have already used this cell sorter configuration to locate thymocytes of the outer cortex of the murine thymus and then to analyse them for two surface markers. This was done by dipping the intact thymus in Hoechst 33342, a DNA stain for viable cells then to stain the resultant call suspension for markers conjugated to Fluorescein and Phycoerythrin. On analysis, data was collected on the blue-fluorescing, Hoechst positive cells. Our current use of this dual laser technology centers around a Coumarin-Avidin conjugate which can be bound to an antibody-biotin conjugate and used in conjunction with directly fluoresceinated and phycoerythrin-conjugated antibodies to look at populations which can only be distinguished by using three antibodies in concert. Coumarin is a new fluorescent labeling agent (7-amino-4-methyl coumarin acetic acid AMCA) which emits in the blue region on activation with UV light.
Multiple two and three colour stainings of all combinations of the antibodies used show consistency of established staining profiles. Good conservation of the patterns of antibody staining are observed after interchange of Coumarin conjugates with conventional fluorochromes in several different staining protocols, showing clearly that three colour immunofluorescent analysis is possible in the absence of a Dye laser.