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Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements.
Pokorna, Sarka; Ventura, Ana E; Santos, Tânia C B; Hof, Martin; Prieto, Manuel; Futerman, Anthony H; Silva, Liana C.
Afiliación
  • Pokorna S; Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel; J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejskova 3, 182 23 Prague, Czech Republic. Electronic address: sarka.pokorna@jh-inst.cas.cz.
  • Ventura AE; Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel; iMed.ULisboa - Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; iBB-Institute for Bioengineering and Biosciences, Instituto Supe
  • Santos TCB; iMed.ULisboa - Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, Lisboa, Portugal.
  • Hof M; J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejskova 3, 182 23 Prague, Czech Republic.
  • Prieto M; iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, Lisboa, Portugal; Associate Laboratory i4HB-Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
  • Futerman AH; Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel.
  • Silva LC; iMed.ULisboa - Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. Electronic address: lianacsilva@ff.ulisboa.pt.
J Photochem Photobiol B ; 228: 112404, 2022 Mar.
Article en En | MEDLINE | ID: mdl-35196617
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Colorantes Fluorescentes / Análisis de Datos Idioma: En Revista: J Photochem Photobiol B Asunto de la revista: BIOLOGIA Año: 2022 Tipo del documento: Article Pais de publicación: Suiza
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Colorantes Fluorescentes / Análisis de Datos Idioma: En Revista: J Photochem Photobiol B Asunto de la revista: BIOLOGIA Año: 2022 Tipo del documento: Article Pais de publicación: Suiza