Merck

Assessing Autophagy in Muscle Stem Cells.

Frontiers in cell and developmental biology (2021-02-09)
Silvia Campanario, Ignacio Ramírez-Pardo, Xiaotong Hong, Joan Isern, Pura Muñoz-Cánoves
ABSTRACT

The skeletal muscle tissue in the adult is relatively stable under normal conditions but retains a striking ability to regenerate by its resident stem cells (satellite cells). Satellite cells exist in a quiescent (G0) state; however, in response to an injury, they reenter the cell cycle and start proliferating to provide sufficient progeny to form new myofibers or undergo self-renewal and returning to quiescence. Maintenance of satellite cell quiescence and entry of satellite cells into the activation state requires autophagy, a fundamental degradative and recycling process that preserves cellular proteostasis. With aging, satellite cell regenerative capacity declines, correlating with loss of autophagy. Enhancing autophagy in aged satellite cells restores their regenerative functions, underscoring this proteostatic activity's relevance for tissue regeneration. Here we describe two strategies for assessing autophagic activity in satellite cells from GFP-LC3 reporter mice, which allows direct autophagosome labeling, or from non-transgenic (wild-type) mice, where autophagosomes can be immunostained. Treatment of GFP-LC3 or WT satellite cells with compounds that interfere with autophagosome-lysosome fusion enables measurement of autophagic activity by flow cytometry and immunofluorescence. Thus, the methods presented permit a relatively rapid assessment of autophagy in stem cells from skeletal muscle in homeostasis and in different pathological scenarios such as regeneration, aging or disease.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
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Triton X-100, laboratory grade
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Fetal Bovine Serum, non-USA origin, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Poly-L-lysine solution, 0.1 % (w/v) in H2O
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Triton X-100, for molecular biology
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Dispase® II, protease
Sigma-Aldrich
Bafilomycin A1 from Streptomyces griseus, ≥90% (HPLC)