Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.

Associate Laboratory i4HB Institute for Health and Bioeconomy at Instituto Superior Técnico Universidade de Lisboa Av Rovisco Pais 1049 001 Lisboa Portugal

Department of Biomolecular Sciences Weizmann Institute of Science Rehovot 76100 Israel

iBB Institute for Bioengineering and Biosciences Instituto Superior Técnico Universidade de Lisboa Av Rovisco Pais Lisboa Portugal

iMed ULisboa Research Institute for Medicines Faculdade de Farmácia Universidade de Lisboa Av Prof Gama Pinto 1649 003 Lisboa Portugal

J Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences Dolejškova 3 182 23 Prague Czech Republic

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