Separation of alveolar macrophages and dendritic cells via autofluorescence: phenotypical and functional...

In a previous study we demonstrated that an increase of monocytes and dendritic cells (MDC) was In the current study, the bright autofluorescence of alveolar macrophages (AMs) was used to separate them efficiently from the MDC. Sorting of freshly isolated BAL cells resulted in a high-autofluorescent fraction, consisting predominantly of AMs, and a low-autofluorescent fraction containing the MDC, lymphocytes, and granulocytes. Thus, a clear separation between suppressive (AM) and stimulating (MDC) activity was obtained as shown in antigen-specific T cell responses. Flow cytometric parameters, density fractionation, and a series of ED monoclonal antibodies raised against rat macrophage antigens showed that both AMs and MDC were diverse populations. After overnight culture, more than 80% of an MDC population with a density range of 1.065-1.079 changed to a lower density (< 1.056) and morphologically developed into DCs with many processes. Concomitantly, monoclonal antibody ED1 expression changed from a granular pattern to a discrete juxtanuclear spot localization.