BACKGROUND:: The 7-ketocholesterol (7KC)-induced cell death has some characteristics of apoptosis and is associated with polar lipid accumulation. So, we investigated the effects of the broad-spectrum caspase inhibitor z-VAD-fmk and of the caspase-2 inhibitor z-VDVAD-fmk on lipid profile evaluated by staining with Nile Red (NR). METHODS:: The 7KC-treated human monocytic U937 cells were cultured in the absence or in the presence of the caspase inhibitors z-VAD-fmk or z-VDVAD-fmk. When staining with NR is performed, neutral and polar lipids have yellow and orange/red emission, respectively, and fluorescence was then analyzed by flow cytometry (FCM) and by confocal laser scanning microscopy (CLSM) combined with subsequent image processing. The 3D-image sequences were obtained by means of CLSM using spectral analysis, and were analyzed by the factor analysis of medical image sequences algorithm to differentiate spectra inside mixed fluorescence emission and get corresponding specific images. RESULTS:: By FCM, comparatively to untreated cells, higher percentages of red fluorescent cells were identified in 7KC-treated cells. Factor curves and images reveal orange and red fluorescence emissions in 7KC-treated cells and show yellow, orange, and red fluorescence emissions in 7KC-treated cells cultured in the presence of z-VAD-fmk or z-VDVAD-fmk. CONCLUSIONS:: Our data support that investigation by FCM and by spectral analysis in CLSM associated with subsequent image processing provides useful tools to determine the effect of caspase inhibitors on lipid content evaluated with NR. They also favor the hypothesis of relationships between caspase activity and polar lipid accumulation. (c) 2007 International Society for Analytical Cytology.