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doi:10.22028/D291-34573
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 |
Dateien zu diesem Datensatz:
Datei | Beschreibung | Größe | Format | |
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cbin.11618.pdf | 2,53 MB | Adobe PDF | Öffnen/Anzeigen |
Diese Ressource wurde unter folgender Copyright-Bestimmung veröffentlicht: Lizenz von Creative Commons