Application of a flow cytometric method using autofluorescence and a tandem fluorescent dye to analyze human alveolar macrophage surface markers

Michael Y. Viksman, Mark C. Liu, Robert P. Schleimer, Bruce S. Bochner

Research output: Contribution to journalArticle

Abstract

Human resident alveolar macrophages (AM) exhibit autofluorescence when excited by light from 488 nm lasers used by most flow cytometers. Because this autofluorescence occurs at peak 540 nm, it obscures fluorescence generated by commonly used immunofluorescent reagent (e.g., antibodies conjugated to fluorescein isothiocyanate (FITC) or R-phycoerythrin (R-PE)) applied for cell surface marker analysis. Therefore, a two color flow cytometric method has been developed that permits the quantitative phenotypic analysis of AM without influence by their natural autofluorescence. In this method, a commercially available preparation of secondary polyclonal antibodies (recognizing primary specific mouse IgG monoclonal antibodies) that are conjugated to a tandem fluorochrome dye (containing R-PE and Cy5) is used. Using this method, the expression of 12 different surface markers on AM obtained from bronchoalveolar lavage (BAL) of 13 subjects was analyzed and compared with their expression on the surface of peripheral blood monocytes. This method will facilitate analysis of surface markers on AM in a variety of disorders.

Original languageEnglish (US)
Pages (from-to)17-24
Number of pages8
JournalJournal of Immunological Methods
Volume172
Issue number1
DOIs
StatePublished - Jun 3 1994

Keywords

  • Alveolar macrophage
  • Flow cytometry
  • Tandem dye

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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