Detection of Cell-Surface Antigens Using Antibody-Conjugated Fluorospheres (ACF): Application for Six-Color Immunofluorescence

• Published in: BioTechniques Vol. 21; no. 3; pp. 498 - 503
• Main Authors: Beavis, Andrew J, Pennline, Kenneth J
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.09.1996; Eaton
• Subjects: AIDS/HIV; Animals; Antibodies, Monoclonal - immunology; Antigens, Differentiation, T-Lymphocyte - analysis; Antigens, Ly - analysis; Antigens, Surface - analysis; Biological and medical sciences; CD4-Positive T-Lymphocytes - immunology; CD8-Positive T-Lymphocytes - immunology; Color; Flow Cytometry - instrumentation; Flow Cytometry - methods; Fluorescein-5-isothiocyanate - chemistry; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Histocompatibility Antigens Class II - analysis; Immunohematology; Immunophenotyping - instrumentation; Immunophenotyping - methods; Leukocyte Common Antigens - analysis; Leukocyte immunology; Lymphocyte Subsets - immunology; Macrophage-1 Antigen - immunology; Mice; Microspheres; Organometallic Compounds - chemistry; Organophosphorus Compounds - chemistry; Phycocyanin - chemistry; Phycoerythrin - chemistry; Spleen - cytology; Spleen - immunology; Thy-1 Antigens - analysis; Performance evaluation; Flow cytometry; Microsphere; Typing; Leukocyte; Fluorogenic reagent; Rodentia; Monoclonal antibody; Cell subpopulation; Cell surface; Antigen; Vertebrata; Mammalia; Mouse; Clinical biology; Simultaneous measurement; Conjugated compound; Detection; Immunofluorescence; Medical application
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/96213rr03

Antibody-conjugated fluorospheres (ACF) were used to phenotype murine leukocytes by flow cytometric analysis. Multicolor immunofluoresence (beyond simultaneous 4-color analysis) is limited by the availability of specific antibody-fluorochrome conjugates and even further restricted by the spectral emission overlap of many of the fluorochromes when used in combination. Fluorospheres possessing unique excitation/emission spectra can provide much needed versatility to existing protocols of multicolor fluorescence. SKY BLUE (647 nm excitation, 730 nm emission) fluorospheres conjugated to CD11b monoclonal antibody were used in combination with the monoclonal antibodies IAd-FITC, L3T4 (CD4)-PE, LYT2 (CD8)-APC, THY1.2 (CD90)-biotin and B220 (CD45R)-RED613 for the simultaneous detection of six distinct cell-surface antigens in a mixed cell population. All fluorescence signals were resolved, and comparison of results from five-, sixand single-color samples indicated that the percentages of cells positive for specific surface antigens were equivalent.