Isolation and identification of exosomes from breast cancer cells

Yu-yang XING, Ya-ni KANG, Ying QI, Meng-lin LUO, Xiao-wei LI, Czajkowsky DAN, Xiao-dong ZHAO

Abstract


Objective: To isolate exosomes from human breast cancer cell line MDA-MB-231 and to analyze their origin and molecular biological characteristics. Methods: Exosomes were isolated from MDA-MB-231 cells by multi-step differential centrifugation and further examined with atomic force microscope. The expression of marker miR (microRNA)-21 was measured by RT-PCR (reverse transcription-PCR), and the expression of marker protein Flotillin-1 protein was validated by Western blotting. Results: The exosomes were spherical or oval-shaped, with a diameter between 30 to 100 nm, and they were positive in the expressions of miR-21 and Flotillin-1 proteins. Conclusion: The multi-step differential centrifugation is a simple method which can be used to isolate exosomes efficiently. Based on the morphological and molecular features, the extracts obtained from MDA-MB-231 cells are exosomes exactly.

DOI:10.3781/j.issn.1000-7431.2013.07.002


Keywords


Breast neoplasms; Exosome; Flotillin-1 protein; miR-21

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