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Detection of Tissue Factor-Positive Extracellular Vesicles by Laser Scanning Confocal Microscopy

Detection of Tissue Factor-Positive Extracellular Vesicles by Laser Scanning Confocal Microscopy

Yohei Hisada, Alyson C Auriemma, Wyeth Alexander, Cihan Ay, Nigel Mackman

Thromb Res. 2017 Feb;150:65-72.

PMID: 28043041

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP150347594	5(6)-Carboxyfluorescein diacetate N-succinimidyl ester		150347-59-4	Price

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