Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry

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Recently, autophagy, which is a degradative process, has drawn attention as an anti-cell death engineering target in addition to apoptosis in recombinant Chinese hamster ovary (rCHO) cell cultures for enhanced production of therapeutic proteins. Appropriate autophagy monitoring methods, that are suitable for long term CHO cell cultures, are necessary in order to investigate the culture conditions that affect the autophagy pathway and to select appropriate engineering targets for autophagy control. Herein, detailed protocols for autophagy monitoring methods based on flow cytometry are provided using the GFP-LC3-overexpressing CHO DG44 host cell line or MDC-like molecules in rCHO cells grown as an adherent culture with serum-containing medium or suspension culture with serum-free medium. Furthermore, combined with the apoptosis detection based on the Annexin V-PS interaction, the simultaneous detection of autophagy and apoptosis is also described. It is anticipated that the protocols described herein will assist in the fast, high throughput monitoring of autophagy that can support other existing autophagy assays. (C) 2011 Elsevier Inc. All rights reserved.
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Issue Date
2012-03
Language
English
Article Type
Review
Keywords

MONODANSYLCADAVERINE MDC; MAMMALIAN AUTOPHAGY; LC3; APOPTOSIS; CULTURE; ASSAY; PROTEIN; MARKER; SYSTEM; DEATH

Citation

METHODS, v.56, no.3, pp.375 - 382

ISSN
1046-2023
DOI
10.1016/j.ymeth.2011.11.006
URI
http://hdl.handle.net/10203/99503
Appears in Collection
BS-Journal Papers(저널논문)
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