Sonali A Gandhi, Shahnaz Parveen, Munirah Alduhailan, Ramesh Tripathi, Nasser Junedi, Mohammad Saqallah, Matthew A Sanders, Peter M Hoffmann, Katherine Truex, James G Granneman, Christopher V Kelly
{"title":"Methods for making and observing model lipid droplets.","authors":"Sonali A Gandhi, Shahnaz Parveen, Munirah Alduhailan, Ramesh Tripathi, Nasser Junedi, Mohammad Saqallah, Matthew A Sanders, Peter M Hoffmann, Katherine Truex, James G Granneman, Christopher V Kelly","doi":"10.1016/j.crmeth.2024.100774","DOIUrl":null,"url":null,"abstract":"<p><p>We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 μm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11133809/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell Reports Methods","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.crmeth.2024.100774","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/5/14 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
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Abstract
We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 μm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.
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