کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5621835 | 1579193 | 2017 | 8 صفحه PDF | دانلود رایگان |
- There are few methods to visualize surface protein on extracellular vesicles (EVs).
- Tissue factor-positive EVs were visualized by laser scanning confocal microscopy (LSCM).
- Tissue factor-positive EVs were detected in plasma samples from pancreatic cancer patients.
IntroductionIncreased levels of tissue factor-positive extracellular vesicles (TFÂ +Â EVs) have been detected in the plasma of patients with various diseases, including cancer and endotoxemia. Levels of TFÂ +Â EVs in plasma samples can be measured by antigen and activity assays. The aim of the present study was to visualize TFÂ +Â EVs by laser scanning confocal microscopy (LSCM).MethodsEVs 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.ResultsTFÂ +Â 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 TFÂ +Â EVs from low TF-expressing Panc-1 cells. Visualization of TFÂ +Â 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.ConclusionLSCM enables visualization of TFÂ +Â EVs in the supernatant from cultured cells and in clinical samples.
Journal: Thrombosis Research - Volume 150, February 2017, Pages 65-72