We recently introduced a MαCD-based method to efficiently replace virtually the entire population of plasma membrane outer leaflet phospholipids and sphingolipids of cultured mammalian cells with exogenous lipids (Li et al, (2016) Proc. Natl. Acad. Sci USA 113:14025–14030). Here, we show if the lipid-to- MαCD ratio is too high or low, cells can round up and develop membrane leakiness. We found that this cell damage can be reversed/prevented if cells are allowed to recover from the exchange step by incubation in complete growth medium. After exchange and transfer to complete growth medium cell growth was similar to that of untreated cells. In some cases, cell damage was also prevented by carrying out exchange at close to room temperature (rather than at 37°C). Exchange with lipids that do (sphingomyelin) or do not (unsaturated phosphatidylcholine) support a high level of membrane order in lipid vesicles had the analogous effect on plasma membrane order, confirming exogenous lipid localization in the plasma membrane. Importantly, changes in lipid composition and plasma membrane properties after exchange and recovery persisted for several hours. Thus, it should be possible to use lipid exchange to investigate the effect of plasma membrane lipid composition upon several aspects of membrane structure and function.

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.