流式细胞术监测中国仓鼠卵巢细胞自噬

niwanmao

全文链接：http://www.sciencedirect.com/science/article/pii/S1046202311002386
Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry

Jae Seong Lee, Gyun Min Lee

Department of Biological Sciences, Graduate School of Nanoscience & Technology (WCU), KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea

Available online 28 November 2011.

AbstractRecently, 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.




Keywords: Flow cytometry; Autophagy; GFP-LC3; CHO cells