Determination of cholesterol content in NPC cells by cellular ELISA. (A) Binding of GST-PFO to cholesterol increases proportionally to the number of cells in the sample within the range of 1-15×103 cells/well. Triton X-100-permeabilized NPC fibroblasts were exposed to 5 μg/ml GST-PFO followed by anti-GST IgY-peroxidase. Absorbance of the product of peroxidase activity is shown. (B) Permeabilization of cells with Triton X-100 does not extract cholesterol from deposits. Fixed NPC cells were treated with 0.03%-0.2% Triton-X-100 (TX) or 0.05% digitonin, as indicated, and processed for cellular ELISA using GST-PFO. In a series of experiments, prior to labeling with GST-PFO, cells were incubated with 3 mM methyl-β (CDX) for 60 min at 37°C. In these conditions prominent reduction in the binding of GST-PFO to cells is observed. (C) Significant binding of GST-PFO to NPC fibroblasts. NPC cells and healthy controls were processed for cellular ELISA. When indicated, prior to labeling with GST-PFO cells were incubated with 3 mM methyl-β-cyclodextrin. The results are presented as a ratio of absorbance at 450 nm and 595 nm. Data are means ± SEM from four experiments. (D-D’) In non-permeabilized cells cholesterol deposits are not stained with GST-PFO (D) but are labeled with filipin (D’). (D”): merged images of D and D’. Scale bar, 20 μm.