The Novel Methods for Analysis of Exosomes Released from Endothelial Cells and Endothelial Progenitor Cells.
Exosomes (EXs) are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by using nanoparticle tracking analysis (NTA) system. The sensitivities of the cell origin markers for ECs (CD105, CD144) and EPCs (CD34, KDR) were evaluated. The sensitivity and specificity were determined by using positive and negative markers for EXs (CD63), platelets (CD41), erythrocytes (CD235a), and microvesicles (Annexin V). Moreover, the methods were further validated in particle-free plasma and patient samples. Results showed that anti-CD105/anti-CD144 and anti-CD34/anti-KDR had the highest sensitivity and specificity for isolating and detecting EC-EXs and EPC-EXs, respectively. The methods had the overall recovery rate of over 70% and were able to detect the dynamical changes of circulating EC-EXs and EPC-EXs in acute ischemic stroke. In conclusion, we have developed sensitive and specific microbeads/Q-dots fluorescence NTA methods for EC-EX and EPC-EX isolation and detection, which will facilitate the functional study and biomarker discovery.
Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Jinju
J
Wang
Department of Cardiology, The Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, China.
Runmin
R
Guo
College of Health Science, Wuhan Sports University, Wuhan, Hubei 430079, China.
Yi
Y
Yang
Departments of Neurology and Internal Medicine, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Bradley
B
Jacobs
Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China.
Suhong
S
Chen
Department of Neurology, Ochsner Medical Center, Jefferson, LA 70121, USA.
Ifeanyi
I
Iwuchukwu
Departments of Neurology and Internal Medicine, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Kenneth J
KJ
Gaines
Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA; Department of Cardiology, The Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, China; Departments of Neurology and Internal Medicine, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Yanfang
Y
Chen
Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Richard
R
Simman
Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China.
Guiyuan
G
Lv
Department of Cardiology, The Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, China.
Keng
K
Wu
Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Ji C
JC
Bihl