Multicolor Immunofluorescence Analysis

  • D. Recktenwald
Part of the Springer Laboratory book series (SLM)


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.


Antibody Conjugate Flow Cytometry Method Antibody Reagent Excitation Laser Power Significant Technical Difficulty 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Glazer AN. Stryer L. Phycobiliprotein-avidin and phycobiliprotein-biotin conjugates. Methods Enzymol. 1990. 184. P 188–94.PubMedCrossRefGoogle Scholar
  2. Glazer AN. Stryer L. Fluorescent tandem phycobiliprotein conjugates. Emission wavelength shifting by energy transfer. Biophys J. 1983 Sep. 43(3). P 383–6.PubMedCrossRefGoogle Scholar
  3. Lanier LL. Recktenwald DJ. Multicolor immunofluorescence and flow cytometry Methods: a companion to methods in enzymology. 1991 June. 2(3). P 192–199CrossRefGoogle Scholar
  4. Loken MR. Parks DR. Herzenberg LA. Two-color immunofluorescence using a fluorescence-activated cell sorter. J Histochem Cytochem. 1977 Jul. 25(7). P 899–907.PubMedCrossRefGoogle Scholar
  5. Oi VT. Glazer AN. Stryer L. Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J Cell Biol. 1982 Jun. 93(3). P 981–6.PubMedCrossRefGoogle Scholar
  6. Rabinovitch P S. Torres RM. Engel D. Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser. J Immunol. 1986 Apr 15. 136(8). P 2769–75.PubMedGoogle Scholar
  7. Recktenwald D. Method for analysis of subpopulations of blood cells. US Patent No. 4,727,020. February 23, 1988.Google Scholar
  8. Recktenwald D. Peridinin chlorophyll complex as fluorescent label. US Patent No. 4,876,190. October 24, 1989.Google Scholar
  9. Recktenwald D. Prezelin B. Chen CH. Kimura J. Biological pigments as fluorescent labels for cytometry. New Technologies in Cytometry and Molecular Biology, Gary C. Salzman, Editor, Proc. SPIE 1206 P 106-111 (1990)Google Scholar
  10. Terstappen LWMM. Shah VO. Conrad MP. Recktenwald D. Loken MR. Discriminating between damaged and intact cells in fixed flow cytometric samples. Cytometry 1988 September. 9(5). P 477–484.PubMedCrossRefGoogle Scholar
  11. Titus JA. Haugland R. Sharrow SO. Segal DM. Texas red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J Immunol Methods. 1982. 50(2). P 193–204.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1992

Authors and Affiliations

  • D. Recktenwald

There are no affiliations available

Personalised recommendations