Tags

Type your tag names separated by a space and hit enter

Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers from Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cell.
Methods Mol Biol. 2017; 1556:51-102.MM

Abstract

Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7+ myogenic progenitors on their surface between the myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are the primary myogenic stem cells in adult muscle. This chapter describes our laboratory protocols for isolating, culturing, and immunostaining intact myofibers from mouse skeletal muscle as a means for studying satellite cell dynamics. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are plated in dishes coated with PureCol collagen (formerly known as Vitrogen) and maintained in a mitogen-poor medium (± supplemental growth factors). Employing such conditions, satellite cells remain at the surface of the parent myofiber while synchronously undergoing a limited number of proliferative cycles and rapidly differentiate. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. These EDL myofibers are routinely plated individually as adherent myofibers in wells coated with Matrigel and maintained in a mitogen-rich medium, conditions in which satellite cells migrate away from the parent myofiber, proliferate extensively, and generate numerous differentiating progeny. Alternatively, these EDL myofibers can be plated as non-adherent myofibers in uncoated wells and maintained in a mitogen-poor medium (± supplemental growth factors), conditions that retain satellite cell progeny at the myofiber niche similar to the FDB myofiber cultures. However, the adherent myofiber format is our preferred choice for monitoring satellite cells in freshly isolated (Time 0) myofibers. We conclude this chapter by promoting the Nestin-GFP transgenic mouse as an efficient tool for direct analysis of satellite cells in isolated myofibers. While satellite cells have been often detected by their expression of the Pax7 protein or the Myf5nLacZ knockin reporter (approaches that are also detailed herein), the Nestin-GFP reporter distinctively permits quantification of satellite cells in live myofibers, which enables linking initial Time 0 numbers and subsequent performance upon culturing. We additionally point out to the implementation of the Nestin-GFP transgene for monitoring other selective cell lineages as illustrated by GFP expression in capillaries, endothelial tubes and neuronal cells. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular, can also be isolated and analyzed using protocols described herein. Collectively, this chapter provides essential tools for studying satellite cells in their native position and their interplay with the parent myofiber.

Authors+Show Affiliations

Department of Biological Structure, School of Medicine, University of Washington, Health Sciences Building, Room G520, 1959 NE Pacific Street, Box 357420, Seattle, WA, 98195, USA.Department of Biological Structure, School of Medicine, University of Washington, Health Sciences Building, Room G520, 1959 NE Pacific Street, Box 357420, Seattle, WA, 98195, USA.Department of Biological Structure, School of Medicine, University of Washington, Health Sciences Building, Room G520, 1959 NE Pacific Street, Box 357420, Seattle, WA, 98195, USA. reuveni@u.washington.edu.

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

28247345

Citation

Stuelsatz, Pascal, et al. "Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers From Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cell." Methods in Molecular Biology (Clifton, N.J.), vol. 1556, 2017, pp. 51-102.
Stuelsatz P, Keire P, Yablonka-Reuveni Z. Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers from Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cell. Methods Mol Biol. 2017;1556:51-102.
Stuelsatz, P., Keire, P., & Yablonka-Reuveni, Z. (2017). Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers from Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cell. Methods in Molecular Biology (Clifton, N.J.), 1556, 51-102. https://doi.org/10.1007/978-1-4939-6771-1_4
Stuelsatz P, Keire P, Yablonka-Reuveni Z. Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers From Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cell. Methods Mol Biol. 2017;1556:51-102. PubMed PMID: 28247345.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers from Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cell. AU - Stuelsatz,Pascal, AU - Keire,Paul, AU - Yablonka-Reuveni,Zipora, PY - 2017/3/2/entrez PY - 2017/3/2/pubmed PY - 2018/2/27/medline KW - 3F-nlacZ-2E KW - Diaphragm KW - Extensor digitorum longus KW - Extraocular muscles KW - Flexor digitorum brevis KW - Isolated myofiber KW - MLC3F-nLacZ KW - Masseter KW - Myf5nLacZ KW - MyoD KW - Myogenin KW - Nestin-GFP KW - Pax7 KW - Satellite cells KW - Skeletal muscle SP - 51 EP - 102 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol Biol VL - 1556 N2 - Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7+ myogenic progenitors on their surface between the myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are the primary myogenic stem cells in adult muscle. This chapter describes our laboratory protocols for isolating, culturing, and immunostaining intact myofibers from mouse skeletal muscle as a means for studying satellite cell dynamics. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are plated in dishes coated with PureCol collagen (formerly known as Vitrogen) and maintained in a mitogen-poor medium (± supplemental growth factors). Employing such conditions, satellite cells remain at the surface of the parent myofiber while synchronously undergoing a limited number of proliferative cycles and rapidly differentiate. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. These EDL myofibers are routinely plated individually as adherent myofibers in wells coated with Matrigel and maintained in a mitogen-rich medium, conditions in which satellite cells migrate away from the parent myofiber, proliferate extensively, and generate numerous differentiating progeny. Alternatively, these EDL myofibers can be plated as non-adherent myofibers in uncoated wells and maintained in a mitogen-poor medium (± supplemental growth factors), conditions that retain satellite cell progeny at the myofiber niche similar to the FDB myofiber cultures. However, the adherent myofiber format is our preferred choice for monitoring satellite cells in freshly isolated (Time 0) myofibers. We conclude this chapter by promoting the Nestin-GFP transgenic mouse as an efficient tool for direct analysis of satellite cells in isolated myofibers. While satellite cells have been often detected by their expression of the Pax7 protein or the Myf5nLacZ knockin reporter (approaches that are also detailed herein), the Nestin-GFP reporter distinctively permits quantification of satellite cells in live myofibers, which enables linking initial Time 0 numbers and subsequent performance upon culturing. We additionally point out to the implementation of the Nestin-GFP transgene for monitoring other selective cell lineages as illustrated by GFP expression in capillaries, endothelial tubes and neuronal cells. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular, can also be isolated and analyzed using protocols described herein. Collectively, this chapter provides essential tools for studying satellite cells in their native position and their interplay with the parent myofiber. SN - 1940-6029 UR - https://www.unboundmedicine.com/medline/citation/28247345/Isolation_Culture_and_Immunostaining_of_Skeletal_Muscle_Myofibers_from_Wildtype_and_Nestin_GFP_Mice_as_a_Means_to_Analyze_Satellite_Cell_ L2 - https://dx.doi.org/10.1007/978-1-4939-6771-1_4 DB - PRIME DP - Unbound Medicine ER -