Identification and analysis of exosomes secreted from macrophages extracted by different methods.
Exosomes were small vesicles secreted by many cells, and they can play an important role in cell signal transductions. Because the diameter of exosomes is about 30-100 nm, it is so difficult to collection them. In this paper, three kinds of exosomes purifying methods (density gradient ultracentrifugation method, the ultracentrifugation and ultrafiltration method, ExoQuick™ Extraction kit method) were used to collected exosomes in culture supernatants of macrophages. The morphologies of three kinds of exosomes were analyzed by transmission electron microscopy (TEM), and the characteristic molecules such as CD86, LAMP-1, HSP-70 on exosomes were analyzed with Western blot. In addition, the biological activities of exosomes purified by three kinds of methods in vitro were analyzed by ELISA and flow cytometry methods. All experimental results show that the purity and quality of exosomes collected by ExoQuick™ extraction kit and ultracentrifugation and ultrafiltration method were better, and they could enhance the expression of MHC-I in macrophages and promote cells to secrete more TNF-α to cause inflammatory response of macrophages. The analysis pointed out that the advantages and disadvantages of the three methods by biological activities or components of exosomes. Therefore, the ultracentrifugation and ultrafiltration method or the ExoQuick™ Extraction kit method were more suitable to be applied in the scientific research.
Department of Clinical Laboratory, Kunshan First People's Hospital, Affiliated to Jiang Su University Kunshan 215300, China.
Jianjun
J
Wang
Department of Clinical Laboratory, Kunshan First People's Hospital, Affiliated to Jiang Su University Kunshan 215300, China.
Yongliang
Y
Yao
Department of Clinical Laboratory, Kunshan First People's Hospital, Affiliated to Jiang Su University Kunshan 215300, China.
Jianhong
J
Wu
Department of Pathology, Chong Qing Cancer Institute Chong Qing 400030, China.
Guangxin
G
Li
D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D003470:Culture Media / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism* / Q000648:ultrastructure; D005374:Filtration / Q000379:methods*; D005434:Flow Cytometry; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000097382:CD68 Molecule; D052118:Lysosomal-Associated Membrane Protein 1
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; D003470:Culture Media; D018840:HSP70 Heat-Shock Proteins; D051907:Lysosomal Membrane Proteins; D014409:Tumor Necrosis Factor-alpha; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1
Exosomes; MHC-I; TNF-α; macrophage