APPLICATION OF A FLOW CYTOMETRIC METHOD USING AUTOFLUORESCENCE AND A TANDEM FLUORESCENT DYE TO ANALYZE HUMAN ALVEOLAR MACROPHAGE SURFACE-MARKERS

Human resident alveolar macrophages (AM) exhibit autofluorescence when excited by light from 488 nm lasers used by most flow cytometers. Bec ause this autofluorescence occurs at peak 540 nm, it obscures fluoresc ence generated by commonly used immunofluorescent reagents (e.g., anti bodies conjugated to fluorescein isothiocyanate (FITC) or R-phycoeryth rin (R-PE)) applied for cell surface marker analysis. Therefore, a two color flow cytometric method has been developed that permits the quan titative phenotypic analysis of AM without influence by their natural autofluorescence. In this method, a commercially available preparation of secondary polyclonal antibodies (recognizing primary specific mous e IgG monoclonal antibodies) that are conjugated to a tandem fluorochr ome dye (containing R-PE and Cy5) is used. Using this method, the expr ession of 12 different surface markers on AM obtained from bronchoalve olar lavage (BAL) of 13 subjects was analyzed and compared with their expression on the surface of peripheral blood monocytes. This method w ill facilitate analysis of surface markers on AM in a variety of disor ders.