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Cytotechnology

, Volume 52, Issue 2, pp 87–97 | Cite as

Optimized and efficient preparation of astrocyte cultures from rat spinal cord

  • Hao Yang
  • Zhe Liang
  • Jingwen Li
  • Xiping Cheng
  • Na Luo
  • Gong JuEmail author
Original paper

Abstract

Astrocytes constitute a major class of glial cells in the CNS, and play crucial roles in physiological functioning, performance and maintenance of the CNS, as well as promotion of neuronal migration and maturation. Astrocytes have also been directly and indirectly implicated in the pathophysiology of various trauma occurrences, development of neurodegenerative diseases and nerve regeneration. To further understand mechanisms by which astrocytes elicit these effects, the first critical step in the study of astrocytes is the preparation of purified astrocytes cultures. Here we describe a simple and convenient procedure for producing rat primary astrocyte cultures of high purity, viability and proliferation. For astrocyte culture, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, seeding density and composition of the culture medium. Using immunofluorescent antibodies against GFAP and OX-42 in combination of Hoechst 33342 fluorescent staining, we found that the purity of the astrocyte cultures was >99%. Astrocytes had high viability as measured by 3-(4, 5-dimethyl-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, flow cytometric analysis was used to measure and observe variations in the cell cycle after 1–2 passages and proliferation of astrocytes was detected with a high percentage of cells stand in S+G2/M phase. Therefore, the method described here is ideal for experiments, which require highly pure astrocyte cultures.

Keywords

Astrocyte Cell culture Spinal cord Immunofluorescence EGF  l-valine Glutamate Sorbitol 

Notes

Acknowledgements

This work was supported by the National Natural science foundation of China (30230450), Basic Research Program of China (2002CB515301).

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Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Hao Yang
    • 1
  • Zhe Liang
    • 1
  • Jingwen Li
    • 1
  • Xiping Cheng
    • 1
  • Na Luo
    • 1
  • Gong Ju
    • 1
    Email author
  1. 1.The Institute of NeuroscienceThe Fourth Military Medical UniversityXi’anP.R. China

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