Quantification of autophagy flux in isolated mouse skeletal muscle fibers with overexpression of fluorescent protein mCherry-EGFP-LC3.
Quantification of autophagy flux in isolated mouse skeletal muscle fibers with overexpression of fluorescent protein mCherry-EGFP-LC3.
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DOI：
10.1016/j.xpro.2022.101871
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发表时间：
2023-03-17
期刊：
影响因子：
--
通讯作者：
Ma, Jianjie
中科院分区：
文献类型：
--
作者：
Zhou, Xinyu;Cho, Ju Hwan;Yi, Jianxun;Choi, Kyounghan;Park, Ki Ho;Zhu, Hua;Cai, Chuanxi;Haggard, Erin;Zhou, Jingsong;Ko, Jae-Kyun;Ma, Jianjie
由于mccherry - egfp - lc3沿z线的基线表达，对肌纤维的自噬通量的评估可能具有挑战性。我们制定了一项协议来克服这个困难。我们通过电穿孔在成年小鼠FDB肌肉中过表达mcherry - egfp - lc3。然后，我们用酶消化FDB肌肉，产生用于活细胞成像的单个纤维。最后，我们开发了一个基于imagej的程序来消除基线条纹模式，并半自动量化自噬体（APs）和自噬体（ALs），用于自噬通量分析。mCherry-EGFP-LC3在骨骼肌中表达，用于监测自噬活性。分离肌纤维中的自噬小体和自噬小体通过活细胞成像FFT半自动图像处理进行记录，用于定量评估自噬通量。由于mccherry - egfp - lc3沿z线的基线表达，对肌纤维的自噬通量的评估可能具有挑战性。我们制定了一项协议来克服这个困难。我们通过电穿孔在成年小鼠FDB肌肉中过表达mcherry - egfp - lc3。然后，我们用酶消化FDB肌肉，产生用于活细胞成像的单个纤维。最后，我们开发了一个基于imagej的程序来消除基线条纹模式，并半自动量化自噬体（APs）和自噬体（ALs），用于自噬通量分析。
Evaluation of autophagy flux could be challenging for muscle fibers due to the baseline expression of mCherry-EGFP-LC3 along the Z-line. We established a protocol to overcome this difficulty. We overexpress mChery-EGFP-LC3 in the FDB muscle of an adult mouse via electroporation. Then, we enzymatically digest FDB muscle to yield individual fibers for live cell imaging. Finally, we develop an ImageJ-based program to eliminate the baseline striation pattern and semi-automatically quantify autophagosomes (APs) and autolysosomes (ALs) for autophagy flux analysis. mCherry-EGFP-LC3 is expressed in skeletal muscle for monitoring autophagy activity Autophagosomes and autolysosomes in isolated muscle fibers are recorded by live cell imaging FFT based semi-automatic image processing for quantitative evaluation of autophagy flux Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Evaluation of autophagy flux could be challenging for muscle fibers due to the baseline expression of mCherry-EGFP-LC3 along the Z-line. We established a protocol to overcome this difficulty. We overexpress mChery-EGFP-LC3 in the FDB muscle of an adult mouse via electroporation. Then, we enzymatically digest FDB muscle to yield individual fibers for live cell imaging. Finally, we develop an ImageJ-based program to eliminate the baseline striation pattern and semi-automatically quantify autophagosomes (APs) and autolysosomes (ALs) for autophagy flux analysis.
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DOI：
10.1002/cpz1.89
发表时间：
2021-05
期刊：
Current protocols
影响因子：
--
作者：
Dobson ETA;Cimini B;Klemm AH;Wählby C;Carpenter AE;Eliceiri KW
通讯作者：
Eliceiri KW
影响因子：
4.6
作者：
Kondratskyi A;Kondratska K;Vanden Abeele F;Gordienko D;Dubois C;Toillon RA;Slomianny C;Lemière S;Delcourt P;Dewailly E;Skryma R;Biot C;Prevarskaya N
通讯作者：
Prevarskaya N
影响因子：
13.6
作者：
Li, Ling;Wang, Zhao V.;Lin, Fangming
通讯作者：
Lin, Fangming
影响因子：
9.3
作者：
Xiao Y;Karam C;Yi J;Zhang L;Li X;Yoon D;Wang H;Dhakal K;Ramlow P;Yu T;Mo Z;Ma J;Zhou J
通讯作者：
Zhou J
影响因子：
3.7
作者：
Tjondrokoesoemo A;Park KH;Ferrante C;Komazaki S;Lesniak S;Brotto M;Ko JK;Zhou J;Weisleder N;Ma J
通讯作者：
Ma J