Multicolor Immunofluorescence Analysis
For many research questions it is desirable to measure subsets of cell populations that have been identified by immunofluorescence. In principle this could be achieved by sorting positive and negative populations of the cells of interest, dissociating and washing out the first reagent, and then staining the two populations with another antibody reagent labeled with the same fluorophor for subsequent analysis. However, this approach is rarely taken because the availability of many different dyes suitable as labels for immunofluorescence makes the simultaneous measurement of many subpopulations in one sample possible. The first simultaneous two-color/one-laser immunofluorescence method for flow cytometry used fluorescein isothiocyanate (FITC) and rhodamine conjugates with suboptimal 514-nm excitation from an argon ion laser (Loken et al. 1977). Subsequently, FITC and Texas red conjugates were used with two-laser excitation (e. g., Titus et al. 1982). The development of phycoerythrin (PE) antibody conjugates made two-color immunofluorescence (FITC/PE) with single-wavelength excitation at 488 nm a routine method (Oi et al. 1982; Glazer et al. 1990). The more recent development of energy transfer complex conjugates, using PEs as donor and phycobiliproteins or synthetic dyes as acceptors (Glazer et al. 1983; Recktenwald et al. 1991; Ernst et al. in prep.) and the discovery that peridinin chlorophyll (PerCP) proteins are stable when conjugated to immunoglobulins and show acceptable nonspecific binding behavior (Recktenwald 1990; Recktenwald et al. 1991) allow three-color immunofluorescence analysis without significant technical difficulties today. A comprehensive list of dyes for multicolor immunofluorescence can be found in the references (Recktenwald et al. 1991; Lanier et al. 1991). Methods that combine immunofluorescence with DNA staining are not reviewed here. An example is described in Rabinovitch et al. 1986.
KeywordsAntibody Conjugate Flow Cytometry Method Antibody Reagent Excitation Laser Power Significant Technical Difficulty
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