{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T02:00:14Z","timestamp":1785204014588,"version":"3.55.0"},"posted":{"date-parts":[[2026,7,23]]},"group-title":"Cell Biology","reference-count":39,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T00:00:00Z","timestamp":1784764800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100015326","name":"NIH Common Fund","doi-asserted-by":"crossref","award":["R35GM138156"],"award-info":[{"award-number":["R35GM138156"]}],"id":[{"id":"10.13039\/100015326","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,23]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  <jats:italic>Hydractinia symbiolongicarpus<\/jats:italic>\n                  is a powerful model for stem cell research and maintains a population of pluripotent adult stem cells throughout its lifetime. Here we describe a gene expression-agnostic FACS technique to isolate a live cell population from\n                  <jats:italic>Hydractinia<\/jats:italic>\n                  feeding polyps that appear to be stem cells. This technique utilizes only the general cellular component stains DAPI, DRAQ5, Calcein AM, and Pyronin Y. The stem cell population was identified via subtractive gating based on samples whose stem cell populations had been selectively depleted with the DNA-alkylating agent Mitomycin C. To validate the identity of the isolated population, a colorimetric cytological assay capable of simultaneously discriminating between all major\n                  <jats:italic>Hydractinia<\/jats:italic>\n                  cell types in a live-dissociated cell solution was developed using May-Gr\u00fcnwald and Giemsa stains. The isolated cell population was significantly depleted by Mitomycin C administration, had a high RNA content, was proliferative, had a cytological profile that matched that of Piwi1\n                  <jats:sup>+<\/jats:sup>\n                  stem cells, and was \u223c10x enriched with Piwi1\n                  <jats:sup>+<\/jats:sup>\n                  stem cells compared to whole cell suspension, all of which support the conclusion that the isolated population is indeed comprised of stem cells. This gene-agnostic FACS technique will serve future research into\n                  <jats:italic>Hydractinia<\/jats:italic>\n                  stem cell biology by enabling the use of isolated populations of live stem cells in transplantation, cell culture, and spheroid experimentation, and may serve as a reference for the development of new methods in other cnidarian species.\n                <\/jats:p>","DOI":"10.64898\/2026.07.22.740159","type":"posted-content","created":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T14:55:17Z","timestamp":1784818517000},"source":"Crossref","is-referenced-by-count":0,"title":["A gene-agnostic FACS technique to isolate stem cells in\n                  <i>Hydractinia symbiolongicarpus<\/i>\n                  validated with cytology"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4878-1961","authenticated-orcid":false,"given":"Zachary M.","family":"Lane","sequence":"first","affiliation":[{"name":"Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL 32080"},{"name":"Department of Biology, University of Florida, Gainesville, FL, 32611, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5001-6524","authenticated-orcid":false,"given":"Christine E.","family":"Schnitzler","sequence":"additional","affiliation":[{"name":"Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL 32080"},{"name":"Department of Biology, University of Florida, Gainesville, FL, 32611, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026072718501495000_2026.07.22.740159v2.1","doi-asserted-by":"publisher","DOI":"10.18637\/jss.v067.i0"},{"key":"2026072718501495000_2026.07.22.740159v2.2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.05506"},{"key":"2026072718501495000_2026.07.22.740159v2.3","doi-asserted-by":"publisher","DOI":"10.1534\/genetics.167.1.357"},{"key":"2026072718501495000_2026.07.22.740159v2.4","doi-asserted-by":"crossref","unstructured":"Chrysostomou, E. , Flici, H. , Gornik, S.G. , Salinas-Saavedra, M. , Gahan, J.M. , McMahon, E.T. , Thompson, K. , Hanley, S. , Kilcoyne, M. , Schnitzler, C.E. , et al. (2022). A cellular and molecular analysis of SoxB-driven neurogenesis in a cnidarian. Elife 11.","DOI":"10.7554\/eLife.78793"},{"key":"2026072718501495000_2026.07.22.740159v2.5","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1186\/s13007-015-0085-3","article-title":"Optimisation approaches for concurrent transmitted light imaging during confocal microscopy","volume":"11","year":"2015","journal-title":"Plant Methods"},{"key":"2026072718501495000_2026.07.22.740159v2.6","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/12.12.1035"},{"key":"2026072718501495000_2026.07.22.740159v2.7","doi-asserted-by":"crossref","unstructured":"Curantz, C. , Krasovec, G. , Horkan, H.R. , Ryan, L. , Varley, \u00c1. , and Frank, U. (2025). An unexpected mode of whole-body regeneration from reaggregated cell suspension in Hydractinia (Cnidaria, Hydrozoa). 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Zugleich ein beitrag zur kenntnis des baues und der lebenserscheinungen dieser gruppe (Jena: Fischer).","DOI":"10.5962\/bhl.title.9093"},{"key":"2026072718501495000_2026.07.22.740159v2.38","doi-asserted-by":"publisher","DOI":"10.1186\/s13287-019-1165-5"},{"key":"2026072718501495000_2026.07.22.740159v2.39","doi-asserted-by":"publisher","DOI":"10.1084\/jem.159.3.679"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.07.22.740159","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T01:50:26Z","timestamp":1785203426000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.07.22.740159"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,23]]},"references-count":39,"URL":"https:\/\/doi.org\/10.64898\/2026.07.22.740159","relation":{},"subject":[],"published":{"date-parts":[[2026,7,23]]},"subtype":"preprint"}}