Identification and analysis of macrophage-derived foam cells from human atherosclerotic lesions by using a "mock" FL3 channel in flow cytometry

Macrophages are one of the major cell types in atherosclerotic lesions. They are believed to play an important role in the pathogenesis and development of the lesion, but their functional state and phenotypic characteristics are not well understood. Using flow cytometry, we analyzed surface markers of macrophages extracted from tissue digests. However, conventional techniques were hampered by the abundance of cell debris and extracellular lipids, which co-localized with double-positive cells in all fluorescent plots. We therefore developed a method to overcome this problem by using a novel gating technique in multiparameter flow cytometry. This method utilized the third fluorescence channel (FL3) as a "mock" channel, since no antibody conjugated to an FL3-specific fluorochrome was added to the samples. Cells single-positive for macrophage-specific monoclonal antibodies (mAb) conjugated to phycoerythrin (PE) (FL2) were separated from non-specific fluorescent particles in the FL2 versus FL3 fluorescent plot and a region excluding debris could be set. This was then used as a gate to exclude debris also in the first fluorescence channel (FL1) vs. FL2 plot in which expression of a panel of activation markers identified by fluorescein isothiocyanate (FITC)-conjugated mAb was analyzed. Using this strategy, we were able to identify and analyze the phenotype of macrophages from human atherosclerotic lesions. We were also able to sort these cells and in this way obtained a preparation of macrophage-derived foam cells from the tissue with little contamination of debris.