{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T16:11:27Z","timestamp":1790093487842,"version":"4.0.1"},"reference-count":31,"publisher":"American Chemical Society (ACS)","issue":"37","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000266","name":"Engineering and Physical Sciences Research Council","doi-asserted-by":"publisher","award":["MR\/K01577X\/1"],"award-info":[{"award-number":["MR\/K01577X\/1"]}],"id":[{"id":"10.13039\/501100000266","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004789","name":"John Fell Fund, University of Oxford","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004789","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MC_UU_12010"],"award-info":[{"award-number":["MC_UU_12010"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MC_UU_12025"],"award-info":[{"award-number":["MC_UU_12025"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MR\/K01577X\/1"],"award-info":[{"award-number":["MR\/K01577X\/1"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002347","name":"Bundesministerium f?r Forschung, Technologie und Raumfahrt","doi-asserted-by":"publisher","award":["FKZ: 13N15713 \/ 13N15717"],"award-info":[{"award-number":["FKZ: 13N15713 \/ 13N15717"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100016019","name":"Freistaat Th?ringen","doi-asserted-by":"publisher","award":["2020 FGZ: FGI 0031"],"award-info":[{"award-number":["2020 FGZ: FGI 0031"]}],"id":[{"id":"10.13039\/100016019","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["2723\/1  2023  44711651"],"award-info":[{"award-number":["2723\/1  2023  44711651"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["316213987  SFB 1278"],"award-info":[{"award-number":["316213987  SFB 1278"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["460889961"],"award-info":[{"award-number":["460889961"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["EXC 2051   390713860"],"award-info":[{"award-number":["EXC 2051   390713860"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["RTG 3014 521747072"],"award-info":[{"award-number":["RTG 3014 521747072"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100024241","name":"EPA Cephalosporin Fund","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100024241","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["104924\/14\/Z\/14"],"award-info":[{"award-number":["104924\/14\/Z\/14"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000769","name":"University of Oxford","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000769","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001320","name":"Wolfson Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001320","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000268","name":"Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["MR\/K01577X\/1"],"award-info":[{"award-number":["MR\/K01577X\/1"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Leibniz Collaborative Excellence Programme","award":["K548\/2023"],"award-info":[{"award-number":["K548\/2023"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,22]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Plasma membrane vesicles (PMVs) are powerful model systems for studying plasma membrane biophysics, but their production typically relies on chemicals, such as dithiothreitol (DTT) and paraformaldehyde (PFA). DTT is detrimental to protein structure and can alter membrane composition, whereas PFA is a well-established fixative that is widely used in biological studies. Taking this into consideration, we introduced a DTT-free method for generating PMVs using PFA alone. Using flow cytometry and fluorescence microscopy, we validated its robustness across multiple cell types. Measurements with the environment-sensitive probe C-Laurdan show that PMVs produced with PFA plus DTT display higher lipid packing compared to those generated with PFA alone, indicating an effect of DTT on membrane order. Furthermore, fluorescence correlation spectroscopy (FCS) measurements reveal reduced mobility of the immune receptor cluster of differentiation 1d (CD1d) on DTT-treated PMVs. During the evaluation of PMVs in coculture experiments, we further identified residual PFA as the source of cellular toxicity, which was successfully eliminated by dialysis. Finally, we demonstrate the biological applicability of the resulting PMVs by assessing T-cell activation, showing that they preserve key physiological properties of the source cells. Overall, our findings suggest that PMVs generated using PFA alone more faithfully preserve the native properties of the source cell plasma membrane than those produced with the conventional PFA plus DTT protocol. Combined with the effective removal of residual PFA by dialysis, this approach expands the potential of PMVs as physiologically relevant model systems for biological and biomedical research.<\/jats:p>","DOI":"10.1021\/acsomega.6c04767","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T21:02:48Z","timestamp":1788469368000},"page":"55647-55657","source":"Crossref","is-referenced-by-count":0,"title":["Paraformaldehyde-Induced\nVesiculation Preserves Native\nMembrane Physiological States Critical for T-Cell Activation"],"prefix":"10.1021","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6061-8350","authenticated-orcid":true,"given":"Dilip","family":"Shrestha","sequence":"first","affiliation":[{"name":"MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford , ,","place":["Oxford, U.K., OX3 9DS"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veerawut","family":"Veerapongchai","sequence":"additional","affiliation":[{"name":"MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford , ,","place":["Oxford, U.K., OX3 9DS"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Eggeling","sequence":"additional","affiliation":[{"name":"MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford , ,","place":["Oxford, U.K., OX3 9DS"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"issue":"17","key":"2026092210575907500_cit1","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.adf6205","article-title":"Coupling of\nprotein condensates to ordered lipid domains determines functional\nmembrane organization","volume":"9","author":"Wang","year":"2023","journal-title":"Sci. Adv."},{"issue":"31","key":"2026092210575907500_cit2","doi-asserted-by":"publisher","first-page":"9640","DOI":"10.1021\/acs.langmuir.2c01205","article-title":"Artificial\nPalmitoylation of Proteins Controls the Lipid Domain-Selective Anchoring\non Biomembranes and the Raft-Dependent Cellular Internalization","volume":"38","author":"Uchida","year":"2022","journal-title":"Langmuir"},{"issue":"2","key":"2026092210575907500_cit3","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1038\/s41580-022-00524-4","article-title":"Regulation of membrane protein structure\nand function by their lipid\nnano-environment","volume":"24","author":"Levental","year":"2023","journal-title":"Nat. Rev. Mol. Cell Biol."},{"issue":"6","key":"2026092210575907500_cit4","doi-asserted-by":"publisher","first-page":"361","DOI":"10.1038\/nrm.2017.16","article-title":"The mystery of membrane\norganization: composition, regulation and roles of lipid rafts","volume":"18","author":"Sezgin","year":"2017","journal-title":"Nat. Rev. Mol. Cell Biol."},{"issue":"6","key":"2026092210575907500_cit5","doi-asserted-by":"publisher","first-page":"1042","DOI":"10.1038\/nprot.2012.059","article-title":"Elucidating membrane structure and protein behavior\nusing giant plasma membrane vesicles","volume":"7","author":"Sezgin","year":"2012","journal-title":"Nat. Protoc."},{"issue":"9","key":"2026092210575907500_cit6","doi-asserted-by":"publisher","first-page":"3165","DOI":"10.1073\/pnas.0611357104","article-title":"Large-scale\nfluid\/fluid phase separation\nof proteins and lipids in giant plasma membrane vesicles","volume":"104","author":"Baumgart","year":"2007","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"51","key":"2026092210575907500_cit7","doi-asserted-by":"publisher","first-page":"22050","DOI":"10.1073\/pnas.1016184107","article-title":"Palmitoylation\nregulates raft affinity for the majority of integral raft proteins","volume":"107","author":"Levental","year":"2010","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"4266","key":"2026092210575907500_cit8","doi-asserted-by":"publisher","first-page":"743","DOI":"10.1126\/science.982044","article-title":"Plasma\nmembrane vesiculation: a new technique for isolation of plasma membranes","volume":"194","author":"Scott","year":"1976","journal-title":"Science"},{"issue":"37","key":"2026092210575907500_cit9","doi-asserted-by":"publisher","first-page":"48982","DOI":"10.1021\/acsami.4c07234","article-title":"Cellular Output and Physicochemical\nProperties of the Membrane-Derived Vesicles Depend on Chemical Stimulants","volume":"16","author":"Shrestha","year":"2024","journal-title":"ACS Appl. Mater. Interfaces"},{"issue":"28","key":"2026092210575907500_cit10","doi-asserted-by":"publisher","first-page":"11411","DOI":"10.1073\/pnas.1105996108","article-title":"Raft domains of variable\nproperties\nand compositions in plasma membrane vesicles","volume":"108","author":"Levental","year":"2011","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"21","key":"2026092210575907500_cit11","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202102109","article-title":"Superelasticity of\nPlasma- and Synthetic\nMembranes Resulting from Coupling of Membrane Asymmetry, Curvature,\nand Lipid Sorting","volume":"8","author":"Steinkuhler","year":"2021","journal-title":"Adv. Sci. (Weinh)"},{"issue":"1","key":"2026092210575907500_cit12","doi-asserted-by":"publisher","first-page":"100155","DOI":"10.1016\/j.jlr.2021.100155","article-title":"Loss of plasma membrane\nlipid asymmetry can induce ordered domain (raft) formation","volume":"63","author":"Kakuda","year":"2022","journal-title":"J. Lipid Res."},{"issue":"11","key":"2026092210575907500_cit13","doi-asserted-by":"publisher","first-page":"865","DOI":"10.1038\/nmeth.f.314","article-title":"Membrane molecules mobile\neven after chemical fixation","volume":"7","author":"Tanaka","year":"2010","journal-title":"Nat. Methods"},{"issue":"1","key":"2026092210575907500_cit14","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1186\/s40580-017-0099-9","article-title":"Mechanical properties of paraformaldehyde-treated\nindividual cells investigated by atomic force microscopy and scanning\nion conductance microscopy","volume":"4","author":"Kim","year":"2017","journal-title":"Nano Converg"},{"issue":"38","key":"2026092210575907500_cit15","doi-asserted-by":"publisher","first-page":"39872","DOI":"10.1074\/jbc.M406912200","article-title":"Glutathione is required to regulate\nthe formation of\nnative disulfide bonds within proteins entering the secretory pathway","volume":"279","author":"Chakravarthi","year":"2004","journal-title":"J. Biol. Chem."},{"issue":"4","key":"2026092210575907500_cit16","doi-asserted-by":"publisher","first-page":"677","DOI":"10.1007\/s13361-015-1319-3","article-title":"S- to N-Palmitoyl Transfer\nDuring Proteomic Sample Preparation","volume":"27","author":"Ji","year":"2016","journal-title":"J. Am.\nSoc. Mass Spectrom."},{"issue":"3","key":"2026092210575907500_cit17","doi-asserted-by":"publisher","first-page":"339","DOI":"10.1093\/jb\/mvab144","article-title":"Basic structure and\ncytocompatibility\nof giant membrane vesicles derived from paraformaldehyde-exposed human\ncells","volume":"171","author":"Okada","year":"2022","journal-title":"J. Biochem."},{"issue":"1","key":"2026092210575907500_cit18","doi-asserted-by":"publisher","first-page":"2200125","DOI":"10.1002\/adtp.202200125","article-title":"Cell-free,\nDendritic Cell-mimicking Extracellular Blebs for Molecularly Controlled\nVaccination","volume":"6","author":"Thone","year":"2023","journal-title":"Adv. Ther (Weinh)"},{"key":"2026092210575907500_cit19","doi-asserted-by":"publisher","first-page":"59","DOI":"10.1016\/j.ab.2018.07.012","article-title":"Wash-free instant detection\nof giant\nplasma membrane vesicles","volume":"557","author":"Okada","year":"2018","journal-title":"Anal. Biochem."},{"issue":"1","key":"2026092210575907500_cit20","doi-asserted-by":"publisher","first-page":"139","DOI":"10.15252\/embj.201695709","article-title":"Glyoxal\nas an alternative fixative to formaldehyde\nin immunostaining and super-resolution microscopy","volume":"37","author":"Richter","year":"2018","journal-title":"EMBO J."},{"key":"2026092210575907500_cit21","doi-asserted-by":"publisher","first-page":"190","DOI":"10.1016\/j.fob.2014.02.003","article-title":"Fixation-induced cell blebbing on spread cells inversely correlates\nwith phosphatidylinositol 4,5-bisphosphate level in the plasma membrane","volume":"4","author":"Zhao","year":"2014","journal-title":"FEBS Open Bio"},{"key":"2026092210575907500_cit22","doi-asserted-by":"publisher","first-page":"129","DOI":"10.1016\/bs.mie.2018.02.007","article-title":"Giant Plasma\nMembrane Vesicles: An\nExperimental Tool for Probing the Effects of Drugs and Other Conditions\non Membrane Domain Stability","volume":"603","author":"Gerstle","year":"2018","journal-title":"Methods Enzymol."},{"issue":"4","key":"2026092210575907500_cit23","doi-asserted-by":"publisher","first-page":"1309","DOI":"10.1073\/pnas.1817498116","article-title":"Chemically\ninduced vesiculation as a platform for studying TMEM16F activity","volume":"116","author":"Han","year":"2019","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"47","key":"2026092210575907500_cit24","doi-asserted-by":"publisher","first-page":"23671","DOI":"10.1073\/pnas.1910097116","article-title":"Sterile activation of invariant natural killer\nT cells by ER-stressed antigen-presenting cells","volume":"116","author":"Bedard","year":"2019","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"7","key":"2026092210575907500_cit25","doi-asserted-by":"publisher","first-page":"1387","DOI":"10.1002\/cphc.201402794","article-title":"Spectral imaging\nto measure heterogeneity in membrane lipid packing","volume":"16","author":"Sezgin","year":"2015","journal-title":"ChemPhysChem"},{"issue":"1","key":"2026092210575907500_cit26","doi-asserted-by":"publisher","first-page":"7426","DOI":"10.1038\/s41598-019-43529-4","article-title":"Generation of a Jurkat-based\nfluorescent reporter cell line to evaluate lipid antigen interaction\nwith the human iNKT cell receptor","volume":"9","author":"Humeniuk","year":"2019","journal-title":"Sci. Rep."},{"issue":"51","key":"2026092210575907500_cit27","doi-asserted-by":"publisher","first-page":"20490","DOI":"10.1073\/pnas.0710145104","article-title":"Modulation\nof human natural killer T cell ligands on TLR-mediated antigen-presenting\ncell activation","volume":"104","author":"Salio","year":"2007","journal-title":"Proc. Natl. Acad. Sci. U. S.\nA."},{"issue":"6","key":"2026092210575907500_cit28","doi-asserted-by":"publisher","first-page":"958","DOI":"10.1093\/bioinformatics\/btv687","article-title":"FoCuS-point: software\nfor STED fluorescence correlation and time-gated single photon counting","volume":"32","author":"Waithe","year":"2016","journal-title":"Bioinformatics"},{"issue":"3","key":"2026092210575907500_cit29","doi-asserted-by":"publisher","first-page":"477","DOI":"10.1083\/jcb.200602085","article-title":"Reassembly\nof contractile actin cortex in cell blebs","volume":"175","author":"Charras","year":"2006","journal-title":"J.\nCell Biol."},{"issue":"13","key":"2026092210575907500_cit30","doi-asserted-by":"publisher","first-page":"134004","DOI":"10.1088\/1361-6463\/aa5dbc","article-title":"Laurdan and\nDi-4-ANEPPDHQ probe different properties of the membrane","volume":"50","author":"Amaro","year":"2017","journal-title":"J. Phys. D Appl. Phys."},{"key":"2026092210575907500_cit31","doi-asserted-by":"publisher","first-page":"756","DOI":"10.3389\/fcell.2020.00756","article-title":"Quantitative Analysis\nof Cellular Lipid Compositions During Acute Proteotoxic ER Stress\nReveals Specificity in the Production of Asymmetric Lipids","volume":"8","author":"Reinhard","year":"2020","journal-title":"Front Cell Dev Biol."}],"container-title":["ACS Omega"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsomega.6c04767","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acsodf\/article-pdf\/11\/37\/55647\/68295033\/acsomega.6c04767.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acsodf\/article-pdf\/11\/37\/55647\/68295033\/acsomega.6c04767.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T15:44:36Z","timestamp":1790091876000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acsodf\/article\/11\/37\/55647\/5414116\/Paraformaldehyde-Induced-Vesiculation-Preserves"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":31,"journal-issue":{"issue":"37","published-online":{"date-parts":[[2026,9,3]]},"published-print":{"date-parts":[[2026,9,22]]}},"URL":"https:\/\/doi.org\/10.1021\/acsomega.6c04767","relation":{},"ISSN":["2470-1343"],"issn-type":[{"value":"2470-1343","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]}}}