Use of Allophycocyanin Allows Quantitative Description by Flow Cytometry of Alveolar Macrophage Surface Antigens Present in Low Numbers of Cells

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Abstract
Autofluorescence has limited quantitative description by flow cytometry of certain alveolar macrophage surface antigens. Autofluorescence is most significant when macrophages are excited by blue light and emission is monitored in the green-yellow light range. Because of recent advances in flow cytometry and in the development of fluorescent labelling compounds, such as allophycocyanin, autofluorescence can be minimized by the use of red excitation and emission wavelengths. We have developed a three-step labelling technique that discretely identifies the minor subpopulation of normal rat alveolar macrophages that express Class II major histocompatibility (Ia) antigens. Using this technique, we observed that alveolar macrophage Ia antigen expression increases at least fourfold after intravenous administration of bacillus Calmette-Guerin to previously primed rats. Additionally, we observed that in vitro treatment of alveolar macrophages by exposure to gamma interferon results in an increase in Ia antigen expression. This new method is significant because flow cytometry is a powerful tool with which to analyze quickly, accurately, and reproducibly alveolar macrophage surface antigens.