High-resolution mapping of phase behavior in a ternary lipid mixture: do lipid-raft phase boundaries depend on the sample preparation procedure?

For some time now, we have been using a fluorescence resonance energy transfer (FRET)-based strategy to conduct high-resolution studies of phase behavior in ternary lipid-raft membrane mixtures. Our FRET experiments can be carried out on ordinary, polydisperse multilamellar vesicle suspensions, so we are able to prepare our samples according to a procedure that was designed specifically to guard against artifactual phase separation. In some respects (i.e., the number and nature of two-phase regions observed), our phase diagrams are consistent with those in previously published reports. However, in other respects (i.e., overall size of miscibility gaps, phase boundary locations and their dependence on temperature), there are clear differences. Here, we present FRET data taken in dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine/cholesterol (DOPC/DPPC/Chol) mixtures at 25.0, 35.0, and 45.0 degrees C. Comparisons between our results and previously reported phase boundaries suggest that lipid-raft mixtures may be particularly susceptible to demixing effects during sample preparation.