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Titel: Flow cytometric quantification of apoptotic and proliferating cells applying an improved method for dissociation of spheroids
VerfasserIn: Metzger, Wolfgang
Rösch, Barbara
Sossong, Daniela
Bubel, Monika
Pohlemann, Tim
Sprache: Englisch
Titel: Cell Biology International
Bandnummer: 45
Heft: 8
Seiten: 1633-1643
Verlag/Plattform: Wiley
Erscheinungsjahr: 2021
Freie Schlagwörter: activated caspase 3
dissociation
flow cytometry
Ki67
live‐dead‐assay
spheroids
DDC-Sachgruppe: 610 Medizin, Gesundheit
Dokumenttyp: Journalartikel / Zeitschriftenartikel
Abstract: Spheroids are a promising tool for many cell culture applications, but their microscopic analysis is limited. Flow cytometry on a single cell basis, which requires a gentle but also efficient dissociation of spheroids, could be an alternative analysis. Mono-culture and coculture spheroids consisting of human fibroblasts and human endothelial cells were generated by the liquid overlay technique and were dissociated using AccuMax as a dissociation agent combined with gentle mechanical forces. This study aimed to quantify the number of apoptotic and proliferative cells. We were able to dissociate spheroids of differing size, age, and cellular composition in a single-step dissociation protocol within 10 min. The number of single cells was higher than 95% and in most cases, the viability of the cells after dissociation was higher than 85%. Coculture spheroids exhibited a higher sensitivity as shown by lower viability, higher amount of cellular debris, and a higher amount of apoptotic cells. Considerable expression of the proliferation marker Ki67 could only be seen in 1-day-old spheroids but was already downregulated on Day 3. In summary, our dissociation protocol enabled a fast and gentle dissociation of spheroids for the subsequent flow cytometric analysis. The chosen cell type had a strong influence on cell viability and apoptosis. Initially high rates of proliferative cells decreased rapidly and reached values of healthy tissue 3 days after generation of the spheroids. In conclusion, the flow cytometry of dissociated spheroids could be a promising analytical tool, which could be ideally combined with microscopic techniques.
DOI der Erstveröffentlichung: 10.1002/cbin.11618
Link zu diesem Datensatz: urn:nbn:de:bsz:291--ds-345739
hdl:20.500.11880/31649
http://dx.doi.org/10.22028/D291-34573
ISSN: 1095-8355
1065-6995
Datum des Eintrags: 26-Aug-2021
Fakultät: M - Medizinische Fakultät
Fachrichtung: M - Chirurgie
Professur: M - Prof. Dr. Tim Pohlemann
Sammlung:SciDok - Der Wissenschaftsserver der Universität des Saarlandes

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