Elsevier

Analytical Biochemistry

Volume 272, Issue 2, 1 August 1999, Pages 135-142
Analytical Biochemistry

Regular Article
Serum-Free Culture Conditions for Analysis of Secretory Proteinases during Myogenic Differentiation of Mouse C2C12 Myoblasts

https://doi.org/10.1006/abio.1999.4163Get rights and content

Abstract

We have been studying extracellular proteins such as proteinases and attachment factors under serum-free culture conditions. A number of studies on myogenesis using an in vitro culture system have reported that proteinases and ECM components play significant roles in muscle differentiation. However, most of the studies were performed in the presence of serum. Serum is abundant in the aforementioned proteins and its use in serum-free culture affects many cellular functions significantly. In this study, we tried to establish serum-free culture conditions for analyzing extracellular proteins involved in mouse myogenic differentiation. By evaluating media, supplements, and procedure of cell inoculation under serum-free conditions and by comparing the resultant conditions with conventional conditions on differentiated characteristics of the cells, it was revealed that serum-free Dulbecco's modified Eagle's medium/Ham's F-12 plus insulin more efficiently supported myogenesis morphologically and biochemically than conventional 2% horse serum-containing culture and that secretory proteinases obtained from our serum-free culture were different from those obtained utilizing conventional serum-free cultures in their activities and patterns. Since our serum-free medium consists of simple components, the medium is low cost and easy to prepare. Furthermore, the results suggest that our culture conditions are superior to conventional conditions biochemically and morphologically and will provide more precise and accurate information on extracellular proteins involved in myogenesis.

References (36)

  • H. Weintraub

    Cell

    (1993)
  • E.N. Olson

    Dev. Biol.

    (1992)
  • F. Miralles et al.

    J. Biol. Chem.

    (1998)
  • B.J. Gilpin et al.

    J. Biol. Chem.

    (1998)
  • K.B. Kwak et al.

    FEBS Lett.

    (1993)
  • N. Dourdin et al.

    Exp. Cell Res.

    (1997)
  • U. Kuhl et al.

    Dev. Biol.

    (1986)
  • R.F. Foster et al.

    Dev. Biol.

    (1987)
  • P. Clark et al.

    Exp. Cell Res.

    (1997)
  • D. Yaffe et al.

    Differentiation

    (1977)
  • K. Ichikawa et al.

    Exp. Cell Res.

    (1993)
  • M. Kumegawa et al.

    Dev. Biol.

    (1980)
  • P. Dollenmeier et al.

    Exp. Cell Res.

    (1981)
  • J.M. Lyles et al.

    Int. J. Dev. Neurosci.

    (1992)
  • Y. Yoshiko et al.

    Life Sci.

    (1996)
  • G. Pegolo et al.

    Int. J. Dev. Neurosci.

    (1990)
  • I. Ii et al.

    Dev. Biol.

    (1982)
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    This study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, and Science of Japan.

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