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dc.contributor.authorAbrahamsen, Irenen_US
dc.contributor.authorLorens, James B.en_US
dc.date.accessioned2013-10-29T15:54:17Z
dc.date.available2013-10-29T15:54:17Z
dc.date.issued2013-08-19eng
dc.PublishedBMC Cell Biology 14:36eng
dc.identifier.issn1471-2121
dc.identifier.urihttps://hdl.handle.net/1956/7454
dc.description.abstractBackground: Tissue microenvironments comprise different extracellular matrix (ECM) proteins that regulate cellular responsiveness to growth factors. In vitro culture of adherent cells on ECM-coated substrata is commonly used to study microenvironmental influence on specific cell signaling responses. Phosphorylation-specific flow cytometry can be utilized to quantify intracellular phosphorylation-dependent signaling events in single cells. However this approach necessitates trypsinization of adherent cells to accommodate flow cytometric analysis. Trypsin is a potent activator of cell signaling and can obscure signal transduction events induced by other factors. Results: To address this we developed a cold trypsin-phosphorylation-specific flow cytometry protocol, where adherent cells are prepared for flow cytometric analysis on ice (~0°C), a temperature where trypsin retains activity but where intracellular kinases are inactive. We show that this straightforward approach can be used to quantify intracellular pERK levels in single adherent primary human vascular smooth muscle cells grown on different ECM. Conclusions: Exploiting the limited temperature dependence of trypsin facilitated development of a generally applicable phosphorylation-specific flow cytometry method for analysis of adherent cell types including primary patient derived cells. We demonstrate the utility of cold trypsin-phosphorylation-specific flow cytometry analysis of cell signaling to measure microenvironmental influence in single adherent cells.en_US
dc.language.isoengeng
dc.publisherBioMed Centraleng
dc.rightsAttribution CC BYeng
dc.rights.urihttp://creativecommons.org/licenses/by/2.0/eng
dc.subjectFlow cytometryeng
dc.subjectPhosphorylationeng
dc.subjectCell signalingeng
dc.subjectExtracellular matrixeng
dc.titleEvaluating Extracellular Matrix influence on adherent cell signaling by Cold Trypsin Phosphorylation-specific Flow Cytometryen_US
dc.typePeer reviewed
dc.typeJournal article
dc.date.updated2013-10-01T19:10:49Z
dc.description.versionpublishedVersionen_US
dc.rights.holderCopyright 2013 Abrahamsen and Lorens; licensee BioMed Central Ltd.
dc.rights.holderIren Abrahamsen et al.; licensee BioMed Central Ltd.
dc.source.articlenumber36
dc.identifier.doihttps://doi.org/10.1186/1471-2121-14-36
dc.identifier.cristin1059000
dc.source.journalBMC Cell Biology
dc.source.4014


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