{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T04:57:26Z","timestamp":1779944246921,"version":"3.53.1"},"reference-count":45,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2024,10,28]],"date-time":"2024-10-28T00:00:00Z","timestamp":1730073600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R44\u2010GM139504"],"award-info":[{"award-number":["R44\u2010GM139504"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R43\u2010GM140527"],"award-info":[{"award-number":["R43\u2010GM140527"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01\u2010EB033155"],"award-info":[{"award-number":["R01\u2010EB033155"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["F31HL158020\u201003"],"award-info":[{"award-number":["F31HL158020\u201003"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["T32GM132089\u201001"],"award-info":[{"award-number":["T32GM132089\u201001"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["5T32GM007226\u201043"],"award-info":[{"award-number":["5T32GM007226\u201043"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2024,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins (FPs), and can destroy chemically\u2010sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi\u2010pass flow cytometry approach to address this long\u2010standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single\u2010cell resolution maintained. Chemically\u2010fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular FPs and methanol\u2010sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cellular analysis without the constraints of conventional one\u2010time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno\u2010oncology, stem cell research, and cell biology.<\/jats:p>","DOI":"10.1002\/cyto.a.24904","type":"journal-article","created":{"date-parts":[[2024,10,28]],"date-time":"2024-10-28T14:18:54Z","timestamp":1730125134000},"page":"838-848","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi\u2010pass acquisition"],"prefix":"10.1002","volume":"105","author":[{"given":"Marissa D.","family":"Fahlberg","sequence":"first","affiliation":[{"name":"LASE Innovation Inc.  Waltham Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Forward","sequence":"additional","affiliation":[{"name":"LASE Innovation Inc.  Waltham Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emane Rose","family":"Assita","sequence":"additional","affiliation":[{"name":"LASE Innovation Inc.  Waltham Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Mazzola","sequence":"additional","affiliation":[{"name":"Center for Regenerative Medicine Massachusetts General Hospital  Boston Massachusetts USA"},{"name":"Harvard Stem Cell Institute  Cambridge Massachusetts USA"},{"name":"Department of Stem Cell and Regenerative Biology Harvard University  Cambridge Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Kiem","sequence":"additional","affiliation":[{"name":"Center for Regenerative Medicine Massachusetts General Hospital  Boston Massachusetts USA"},{"name":"Harvard Stem Cell Institute  Cambridge Massachusetts USA"},{"name":"Department of Stem Cell and Regenerative Biology Harvard University  Cambridge Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maris","family":"Handley","sequence":"additional","affiliation":[{"name":"Center for Regenerative Medicine Massachusetts General Hospital  Boston Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seok\u2010Hyun","family":"Yun","sequence":"additional","affiliation":[{"name":"Wellman Center for Photomedicine Massachusetts General Hospital  Boston Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7880-0401","authenticated-orcid":false,"given":"Sheldon J. 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