Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy

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

Research output: Contribution to journalArticle

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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.

LanguageEnglish (US)
Pages65-72
Number of pages8
JournalThrombosis Research
Volume150
DOIs
StatePublished - Feb 1 2017

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Annexin A5
Thromboplastin
Confocal Microscopy
Anti-Idiotypic Antibodies
Pancreatic Neoplasms
Lipopolysaccharides
Endotoxemia
Cultured Cells
Extracellular Vesicles
Antigens
Cell Line
Neoplasms

Keywords

  • Cancer
  • Extracellular vesicles
  • Laser scanning confocal microscopy
  • Tissue factor

ASJC Scopus subject areas

  • Hematology

Cite this

Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy. / Hisada, Yohei; Auriemma, Alyson C.; Alexander, Wyeth; Ay, Cihan; Mackman, Nigel.

In: Thrombosis Research, Vol. 150, 01.02.2017, p. 65-72.

Research output: Contribution to journalArticle

Hisada, Yohei ; Auriemma, Alyson C. ; Alexander, Wyeth ; Ay, Cihan ; Mackman, Nigel. / Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy. In: Thrombosis Research. 2017 ; Vol. 150. pp. 65-72
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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.",
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N2 - 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.

AB - 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.

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KW - Laser scanning confocal microscopy

KW - Tissue factor

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