title: Evaluating extracellular matrix influence on adherent cell signaling by cold trypsin phosphorylation-specific flow cytometry.
abstract: BACKGROUND
  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.
authors: Department of Biomedicine, University of Bergen, Jonas Lies Vei 91, 5009 Bergen, Norway.
authors: Iren
authors: I
authors: Abrahamsen
authors: James B
authors: JB
authors: Lorens
mesh_terms: D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D003080:Cold Temperature; D005109:Extracellular Matrix / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007797:Laminin; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011651:Pulmonary Artery / Q000166:cytology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014357:Trypsin
chemical_list: D016326:Extracellular Matrix Proteins; D007797:Laminin; D048049:Extracellular Signal-Regulated MAP Kinases; D014357:Trypsin
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