Fig. 3. FRET between Alexa488-IgE (donor) and Cy3-anti-Thy-1 (acceptor) is disrupted by short-chain ceramides. (A) Alexa488-IgE fluorescence monitored as a function of time, showing the effect of sequential addition of Cy3-anti-Thy-1 and 8 µM C₆-cer. Steady-state donor fluorescence intensity values denoted are: I_D, before acceptor addition; I_DA, after acceptor addition; I_DC, after ceramide addition. Typical transfer efficiency (E) between the donor and acceptor was 0.05-0.10. (B) Percent disruption of FRET between Alexa488-IgE and Cy3-anti-Thy-1 by 8 µM C₆-cer (hatched bar), 32 µM C₂-cer (filled bar) and 32 µM C₂-dhcer (open bar). Percent disruption was calculated according to Eqn 1, and averages of at least three experiments plotted, with bars showing s.e.m.