One-week-old human M-CSF differentiated monocyte-derived macrophage cultures were incubated 2 days with 50 μg/ml AcLDL + 5 μm TO9 to induce macrophage deposition of extracellular cholesterol microdomains. Following rinsing, macrophages remained in a one set of cultures (left) and were removed from a second set of cultures (right). Next, both sets of culture wells were incubated 1 day with the indicated additions (20 μg ApoA-I, 100 μg/ml peptide, and 125 μg/ml sphingomyelin complexed with 100 μg/ml peptide 5A) without AcLDL. Cholesterol microdomains were visualized by fluorescence microscopy using anti-cholesterol microdomain Mab 58B1 (green), and nuclei were imaged with DAPI fluorescence staining (blue). To the right of each fluorescence image is the corresponding, phase-contrast microscopic image. AcLDL, acetylated low-density lipoprotein; TO9, TO901317; f/b, followed by; 5A-SPH, 5A-sphingomyelin complex. Bar = 50 μm and applies to all.