{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T18:40:22Z","timestamp":1776883222388,"version":"3.51.2"},"reference-count":39,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T00:00:00Z","timestamp":1776816000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Experimental Animal Research Fund, and Science and Technology Commission of Shanghai Municipality","award":["22140900100"],"award-info":[{"award-number":["22140900100"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Flow cytometric test of erythrocyte osmotic fragility (EOF) is a critical diagnostic tool for evaluating membrane stability in hemolytic disorders and storage lesions of human erythrocytes. However, there is a lack of such protocols tailored for mice, one of the most widely used preclinical animal models. In this work, we developed and optimized a flow cytometric assay of murine EOF, which achieved results comparable to the conventional osmotic fragility test. We also validated the protocol by detecting membrane instability of murine erythrocytes induced by Ca\u00b2\u2009\u207a\u2009-treatment, long-term storage,\u00a0and phenylhydrazine (PHZ)-induced acute anemia,\u00a0respectively. Our results showed that Ca\u00b2\u2009\u207a\u2009-treatment impaired membrane integrity of mouse erythrocytes, resulting in reduced percentage of residual cells compared to the untreated ones (46.5\u2009\u00b1\u20099.5% vs 79.6\u2009\u00b1\u20095.3%,\n                    <jats:italic>P<\/jats:italic>\n                    \u2009&lt;\u20090.0001). We found that murine erythrocytes can be stored in 1 x PBS containing 2% FBS at 4\u00b0C for one week with intact membrane stability (81.6\u2009\u00b1\u20094.6% of residual cells vs 82.1\u2009\u00b1\u20096.0% for the fresh samples, p\u2009&gt;\u20090.05). In PHZ induced anemia model, significant hemolysis was observed at day 3 post injection. In conclusion, our flow cytometric assay of murine EOF is a powerful high-throughput assay for mouse models of membrane disorders, and holds substantial scientific and translational value for investigating pathoetiological mechanisms and developing therapeutic interventions.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0345170","type":"journal-article","created":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T17:46:16Z","timestamp":1776879976000},"page":"e0345170","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["A flow cytometric assay of murine erythrocyte osmotic fragility"],"prefix":"10.1371","volume":"21","author":[{"given":"Pengwei","family":"Zhang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lishuang","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shengbao","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanjuan","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1405-9700","authenticated-orcid":true,"given":"Xianmin","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7529-810X","authenticated-orcid":true,"given":"Ruling","family":"Shen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ying","family":"Lei","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2026,4,22]]},"reference":[{"issue":"3","key":"pone.0345170.ref001","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1159\/000452106","article-title":"Biophysical 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Haematol"},{"issue":"5","key":"pone.0345170.ref008","doi-asserted-by":"crossref","first-page":"899","DOI":"10.1182\/blood.V61.5.899.899","article-title":"Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance","volume":"61","author":"MR Clark","year":"1983","journal-title":"Blood"},{"issue":"3","key":"pone.0345170.ref009","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1002\/(SICI)1096-8652(199807)58:3<206::AID-AJH8>3.0.CO;2-V","article-title":"Cryohemolysis for the detection of hereditary spherocytosis: correlation studies with osmotic fragility and autohemolysis","volume":"58","author":"S Streichman","year":"1998","journal-title":"Am J Hematol"},{"issue":"3","key":"pone.0345170.ref010","first-page":"924","article-title":"Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia","volume":"111","author":"MJ King","year":"2000","journal-title":"Br J Haematol"},{"issue":"5","key":"pone.0345170.ref011","doi-asserted-by":"crossref","first-page":"051501","DOI":"10.1063\/1.4895755","article-title":"Biomechanical properties of red blood cells in health and disease towards microfluidics","volume":"8","author":"G Tomaiuolo","year":"2014","journal-title":"Biomicrofluidics"},{"issue":"4","key":"pone.0345170.ref012","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1016\/j.blre.2013.04.003","article-title":"Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders","volume":"27","author":"L Da Costa","year":"2013","journal-title":"Blood Rev"},{"issue":"6","key":"pone.0345170.ref013","doi-asserted-by":"crossref","first-page":"908","DOI":"10.1177\/1040638720957117","article-title":"Red blood cell osmotic fragility in healthy loggerhead and green sea turtles","volume":"32","author":"R Radisic","year":"2020","journal-title":"J Vet Diagn 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function of the red cell membrane?","volume":"12","author":"N Mohandas","year":"1999","journal-title":"Baillieres Best Pract Res Clin Haematol"},{"issue":"13","key":"pone.0345170.ref020","doi-asserted-by":"crossref","DOI":"10.3390\/ijms25137027","article-title":"Mild Systemic Inflammation Increases Erythrocyte Fragility","volume":"25","author":"CM Stuart","year":"2024","journal-title":"Int J Mol Sci"},{"key":"pone.0345170.ref021","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1016\/j.ebiom.2016.08.006","article-title":"Sickle Cell Trait Increases Red Blood Cell Storage Hemolysis and Post-Transfusion Clearance in Mice","volume":"11","author":"DO Osei-Hwedieh","year":"2016","journal-title":"EBioMedicine"},{"issue":"20","key":"pone.0345170.ref022","doi-asserted-by":"crossref","DOI":"10.1172\/jci.insight.172656","article-title":"Mitapivat reprograms the RBC metabolome and improves anemia in a mouse model of hereditary spherocytosis","volume":"8","author":"A Matte","year":"2023","journal-title":"JCI Insight"},{"issue":"6","key":"pone.0345170.ref023","doi-asserted-by":"crossref","first-page":"1829","DOI":"10.1016\/j.surg.2022.07.016","article-title":"Storage with ethanol attenuates the red blood cell storage lesion","volume":"172","author":"SW Zingg","year":"2022","journal-title":"Surgery"},{"issue":"3","key":"pone.0345170.ref024","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1016\/j.surg.2021.11.020","article-title":"Improving packed red blood cell storage with a high-viscosity buffered storage solution","volume":"171","author":"KE Pulliam","year":"2022","journal-title":"Surgery"},{"issue":"3","key":"pone.0345170.ref025","doi-asserted-by":"crossref","first-page":"666","DOI":"10.1016\/j.surg.2020.07.022","article-title":"Washing packed red blood cells decreases red blood cell storage lesion formation","volume":"169","author":"KE Pulliam","year":"2021","journal-title":"Surgery"},{"issue":"5","key":"pone.0345170.ref026","article-title":"Variability in erythrocyte deformability among various mammals","volume":"236","author":"JE Smith","year":"1979","journal-title":"Am J Physiol"},{"key":"pone.0345170.ref027","doi-asserted-by":"crossref","first-page":"110833","DOI":"10.1016\/j.cbpb.2023.110833","article-title":"Correlation between sphingomyelin and the membrane stability of mammalian erythrocytes","volume":"265","author":"T Yamaguchi","year":"2023","journal-title":"Comp Biochem Physiol B Biochem Mol Biol"},{"issue":"9","key":"pone.0345170.ref028","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0046101","article-title":"Alterations in bone and erythropoiesis in hemolytic anemia: comparative study in bled, phenylhydrazine-treated and Plasmodium-infected mice","volume":"7","author":"R Moreau","year":"2012","journal-title":"PLoS One"},{"issue":"14","key":"pone.0345170.ref029","doi-asserted-by":"crossref","first-page":"7307","DOI":"10.3390\/ijms22147307","article-title":"Minor Changes in Erythrocyte Osmotic Fragility in Trace Amine-Associated Receptor 5 (TAAR5) Knockout Mice","volume":"22","author":"IS Zhukov","year":"2021","journal-title":"Int J Mol Sci"},{"issue":"2","key":"pone.0345170.ref030","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1111\/j.1365-3148.2012.01139.x","article-title":"Red blood cell storage and cell morphology","volume":"22","author":"B Blasi","year":"2012","journal-title":"Transfus Med"},{"issue":"6","key":"pone.0345170.ref031","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1007\/s00277-010-1112-0","article-title":"A prospective study to assess the predictive value for hereditary spherocytosis using five laboratory tests (cryohemolysis test, eosin-5\u2019-maleimide flow cytometry, osmotic fragility test, autohemolysis test, and SDS-PAGE) on 50 hereditary spherocytosis families in Argentina","volume":"90","author":"RL Crisp","year":"2011","journal-title":"Ann Hematol"},{"issue":"1","key":"pone.0345170.ref032","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1002\/cyto.b.21511","article-title":"Flow cytometric osmotic fragility test: Increased assay sensitivity for clinical application in pediatric hematology","volume":"94","author":"O Ciepiela","year":"2018","journal-title":"Cytometry B Clin Cytom"},{"issue":"7","key":"pone.0345170.ref033","doi-asserted-by":"crossref","first-page":"678","DOI":"10.1111\/aor.12826","article-title":"Red Blood Cell Mechanical Fragility Test for Clinical Research Applications","volume":"41","author":"LA Ziegler","year":"2017","journal-title":"Artif Organs"},{"issue":"1","key":"pone.0345170.ref034","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1186\/1751-0147-55-74","article-title":"The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated 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