Cholesterol affects the extent of fusion. (A) Fluorescence images of a CF-labeled RBC fusing to a HAS cell. (a) Frames of unfused cell pair. (b) Low pH–triggered (pH trigger at t = 0, not shown in the picture) HAS–RBC cell pair fusion. Fluorescent dye remains trapped in RBC at the end of acquisition. (c) Fusion of cholesterol-enriched HAS cells. Target RBC membrane completely merged with cholesterol-enriched HAS cell membrane. (B) Comparison of the plateau phase of dye transfer. Entire HAS-RBC cell pairs were included in the ROI and F were compared relative to baseline fluorescence [(F − F₀)/(F_P − F₀)] × 100. F were then compared with the highest intensity of the plateau phase and expressed as percentage relative fluorescence intensity (%RFI). (B, i) Dye transfer in cholesterol-enriched HAS cells. (ii) Dye transfer in untreated HAS cells is slow and RFI increases to only ∼4% compared to cholesterol enriched HAS cells. In i and ii, time zero is the onset of dye transfer. (iii) Relative fluorescence intensity (RFI) measurement of unfused cell pair. (C) Bar chart showing %RFI of cholesterol-enriched HAS cells (with pattern) and control or untreated HAS cells (n > 7).