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Quantification of autophagy flux in isolated mouse skeletal muscle fibers with overexpression of fluorescent protein mCherry-EGFP-LC3
- Source :
- STAR Protocols, Vol 4, Iss 1, Pp 101871- (2023)
- Publication Year :
- 2023
- Publisher :
- Elsevier, 2023.
-
Abstract
- Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
- Subjects :
- Cell Biology
Microscopy
Model Organisms
Science (General)
Q1-390
Subjects
Details
- Language :
- English
- ISSN :
- 26661667
- Volume :
- 4
- Issue :
- 1
- Database :
- Directory of Open Access Journals
- Journal :
- STAR Protocols
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.1cb00a5ad4d34429be28f5b4df5114e4
- Document Type :
- article
- Full Text :
- https://doi.org/10.1016/j.xpro.2022.101871