title: Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy.
abstract: BACKGROUND
  Increased levels of tissue factor-positive extracellular vesicles (TF+EVs) have been detected in the plasma of patients with various diseases, including cancer and endotoxemia. Levels of TF+EVs in plasma samples can be measured by antigen and activity assays. The aim of the present study was to visualize TF+EVs by laser scanning confocal microscopy (LSCM).
  
  
  METHODS
  EVs were isolated from the supernatant of two cultured human pancreatic cancer cell lines (Panc-1 and BxPc-3), from untreated or lipopolysaccharide (LPS) treated whole blood, and from plasma of pancreatic cancer patients. EV-TF activity was determined using an in-house assay. The EVs were labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, which is converted to the impermeant green fluorescent molecule carboxyfluorescein inside the EVs. EVs were either captured using annexin V and detected using a fluorescent-labeled anti-TF antibody, or captured using an anti-TF antibody and detected using fluorescent-labeled annexin V. EVs were visualized by LSCM.
  
  
  RESULTS
  TF+EVs were easily detected from high TF-expressing BxPc-3 cells using annexin V capture, whereas the addition of tyramide amplification was required to detect TF+EVs from low TF-expressing Panc-1 cells. Visualization of TF+EVs in plasma from LPS treated whole human blood and in plasma from pancreatic cancer patients required either capture with annexin V and detection with a fluorescent-labeled anti-TF antibody with tyramide signal amplification, or capture with an anti-TF antibody and detection with a fluorescent-labeled annexin V.
  
  
  CONCLUSIONS
  LSCM enables visualization of TF+EVs in the supernatant from cultured cells and in clinical samples.
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, Thrombosis and Hemostasis Program, UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; K.G. Jensen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø, Norway.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Hisada
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, Thrombosis and Hemostasis Program, UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Alyson C
authors.initials: AC
authors.lastname: Auriemma
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, Thrombosis and Hemostasis Program, UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Wyeth
authors.initials: W
authors.lastname: Alexander
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, Thrombosis and Hemostasis Program, UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Clinical Division of Hematology and Hemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Austria.
authors.forename: Cihan
authors.initials: C
authors.lastname: Ay
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, Thrombosis and Hemostasis Program, UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; K.G. Jensen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø, Norway. Electronic address: nmackman@med.unc.edu.
authors.forename: Nigel
authors.initials: N
authors.lastname: Mackman
mesh_terms: D017304:Annexin A5 / Q000032:analysis; D002460:Cell Line; D045744:Cell Line, Tumor; D000067128:Extracellular Vesicles / Q000473:pathology / Q000648:ultrastructure*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000032:analysis*; D018613:Microscopy, Confocal / Q000379:methods*; D010179:Pancreas / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000097:blood / Q000473:pathology; D013925:Thromboplastin / Q000032:analysis*
chemical_list: D017304:Annexin A5; D018950:Lipopolysaccharide Receptors; D013925:Thromboplastin
keywords: Cancer; Extracellular vesicles; Laser scanning confocal microscopy; Tissue factor