Two- and three-color immunofluorescence using aminocoumarin, fluorescein, and phycoerythrin-labelled antibodies and singel laser flow cytometry

D. Delia, E. Martinez, E. Fontanella, A. Aiello

Research output: Contribution to journalArticlepeer-review


Antibodies coupled to 7-aminocumarin (AMCA) emit a bright blue fluorescence under ultraviolet (UV) excitation and are therefore ideal for three-color immunofluorescence (IF) with fluorescein (FITC) and phycoerythrin (PE) labeled reagents; however, due to the different absorption spectra, the use of these fluorophores for multicolor flow-cytometric analysis requires a double light excitation source (e.g., two-laser system). We report a strategy which uses a single argon-ion laser to simultaneously excite AMCA, FITC, and PE, thus allowing the flow cytometric analysis of three immunological parameters. When the UV-visible argon-ion laser is fitted with an appropriate set of mirrors, the 351.1-363.8 nm (UV) and 488 nm wavelengths (accounting for 80 mW and 520 mW, respectively) are simultaneously generated; these lines can then be exactly focused on the same observation point by an achromatic cylindrical lens. A number of comparative analysis were performed with this instrumental set up to verify the sensitivity of AMCA IF and its possible application for multicolor immunophenotypic evaluation of blood cell subsets. When AMCA- and FITC-labeled antimouse Ig antibodies were assessed for their ability to detect limiting amounts of mouse monoclonal antibody bound to cells, the former was less sensitive than the latter. A number of factors, including differences in excitation energy (80 mW for AMCA and 520 mw for FITC) and extinction coefficients (1.9 x 10 4 for AMCA and 6 x 10 4 for FITC) could explain this result. Single-color IF analysis of blood cells stained with monoclonal antibodies specific for T-cells (CD3, CD4, and CD8) and B-cells (CD19) gave, for each marker, similar results, regardless of the fluorochrome used; similarly, two-color IF using AMCA/FITC, AMCA/PE, and FITC/PE combinations provided comparable results. AMCA fluorescence was minimally detected in the green (requiring only 5% color compensation) but not in the red fluorescence channel. Triple-color detection of CD3, CD4 (or CD8), and CD19 positive blood cell subsets was also achieved. Our data indicate that multiple-color IF analysis of blood T- and B-cell subsets can be performed by flow cytometry and single-laser dual-wavelength excitation using antibodies labeled with AMCA, FITC, and PE. Improvements of the fluorescent reagents and of the optical assembly of the instrument may be required to further increase the resolution of AMCA IF.

Original languageEnglish
Pages (from-to)537-544
Number of pages8
Issue number6
Publication statusPublished - 1991


  • coumarin
  • flow cytometry
  • lymphocytes
  • three-color immunofluorescence

ASJC Scopus subject areas

  • Hematology
  • Cell Biology
  • Pathology and Forensic Medicine
  • Biophysics
  • Endocrinology


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