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GFP and RFP tandemly tagged LC3 (tfLC3) assay

YC Yuan Cao
TS Tao Shen
XH Xiuqing Huang
YL Yajun Lin
BC Beidong Chen
JP Jing Pang
GL Guoping Li
QW Que Wang
SZ Sylvia Zohrabian
CD Chao Duan
YR Yang Ruan
YM Yong Man
SW Shu Wang
JL Jian Li
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The method to evaluate tandem fluorescent LC3 puncta using the mRFP-GFP-LC3 plasmid was previously described [32]. H9c2 cells cultured on cover slips were transduced with the mRFP-GFP-LC3 plasmid for 24 h and then treated with DOX or DOX+APS for another 24 h. Then, the cells were viewed with an inverted fluorescence microscope. If there was a significant population of red-only signals, then the autophagosomes normally matured into autolysosomes (where the GFP is relatively unstable). If most puncta exhibited both red and green signals, autophagy was impaired at some step. Co-localization efficiency of mRFP with GFP signals of tfLC3 puncta was measured using ImageJ software and shown as the percentage of the total number of mRFP puncta.

Copyright and License information:  ©2017 Cao et al.
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