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Fragments or other cell components are similar in size to those of <jats:italic>Mycoplasma<\/jats:italic>; therefore, distinguishing them is difficult. In this study, we used Hoechst staining in combination with carboxyfluorescein succinimidyl ester (CFSE) to label <jats:italic>Mycoplasma<\/jats:italic>. The trigger threshold was set in the Hoechst Blue channel rather than in the default forward scatter channel, utilizing the differences in DNA content between <jats:italic>Mycoplasma<\/jats:italic> and fragments. Subsequently, we identified and isolated it at single\u2010cell resolution via flow cytometry and successfully sorted infectious <jats:italic>Mycoplasma<\/jats:italic> in cell culture. Simultaneously, we validated the accuracy and feasibility of this approach using polymerase chain reaction, fluorescence confocal microscopy, and cryo\u2010electron microscopy. This methodology enabled the diagnosis of <jats:italic>Mycoplasma<\/jats:italic> at extremely low concentrations, significantly enhancing the detection efficiency and facilitating the isolation and purification of parasitic <jats:italic>Mycoplasma<\/jats:italic> in cell culture instead of pure <jats:italic>Mycoplasma<\/jats:italic> culture in artificial media for subsequent studies.<\/jats:p>","DOI":"10.1002\/cyto.a.24908","type":"journal-article","created":{"date-parts":[[2024,12,2]],"date-time":"2024-12-02T10:09:09Z","timestamp":1733134149000},"page":"54-64","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Flow cytometry\u2010based method to detect and separate <i>Mycoplasma hyorhinis<\/i> in cell cultures"],"prefix":"10.1002","volume":"107","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-4512-1590","authenticated-orcid":false,"given":"Chunzhuo","family":"Liu","sequence":"first","affiliation":[{"name":"Institute of Molecular Physiology Shenzhen Bay Laboratory  Guangdong China"},{"name":"Graduate Department Shenzhen Medical Academy of Research and Translation  Guangdong China"},{"name":"Westlake\u2010SMART Program Westlake University  Zhejiang China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-8350-5657","authenticated-orcid":false,"given":"Hui","family":"Wang","sequence":"additional","affiliation":[{"name":"Genomics Core Facility Shenzhen Bay Laboratory  Guangdong China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5772-4793","authenticated-orcid":false,"given":"Shan","family":"Liu","sequence":"additional","affiliation":[{"name":"Bioimaging Core Facility Shenzhen Bay 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