Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy
| Autor: | Yohei Hisada, Nigel Mackman, Wyeth Alexander, Alyson C. Auriemma, Cihan Ay |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Lipopolysaccharide Receptors 030204 cardiovascular system & hematology Cell Line Thromboplastin 03 medical and health sciences Tissue factor Extracellular Vesicles 0302 clinical medicine Antigen Annexin Cell Line Tumor Humans Annexin A5 Pancreas Whole blood Microscopy Confocal biology Chemistry Hematology Cell biology Pancreatic Neoplasms 030104 developmental biology Cell culture biology.protein Antibody |
| Zdroj: | Thrombosis research. 150 |
| ISSN: | 1879-2472 |
| Popis: | Introduction 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. Conclusion LSCM enables visualization of TF + EVs in the supernatant from cultured cells and in clinical samples. |
| Databáze: | OpenAIRE |
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