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Short-term serum supplementation improves glucose-oxygen deprivation in primary cortical cultures grown under serum-free conditions

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Methods in Cell Science

Abstract

Brain ischemia can be studied in vitroby depriving primary neurons of oxygen and glucose by replacing oxygen with argon and glucose with its antimetabolite 2-deoxy-D-glucose. In this contribution, we explain how to construct a reliably functioning ischemia chamber and use it to study neuronal cell death in neuron-enriched fetal primary cortical cultures grown under serum free conditions. We observed that these cultures exhibited a significant cell death even during exposure to oxygenated balanced salt solution used as control for oxygen-glucose deprivation. We show that addition of only 2 fetal calf serum 24 h prior, during, and after treatment almost abolished this undesirable cell loss and proportionally increased cell death induced by oxygen-glucose deprivation. Western blots and immunocytochemistry showed that these effects were mainly due to an increase in neuronal viability under control conditions accompanied by a limited glial proliferation independent of the treatment condition. Under these modified conditions, the cultures could also still be effectively preconditioned by a short-term oxygen-glucose deprivation. In summary, this modified protocol combines the advantages of serum-free neuronal culture, where potentially toxic antimitotic substances can be omitted, with a serum-mediated protection of neurons against unspecific factors and concomitant sensitization for oxygen-glucose deprivation.

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Authors and Affiliations

  1. Research Group Protective Signaling, Zentrum für Molekulare Neurobiologie and Department of Neurology, University Hospital Hamburg-Eppendorf, Hamburg, Germany

    Jan Lewerenz, Susanne Thomsen, Julius A. Steinbeck & Axel Methner

  2. Research Group Protective Signaling, Zentrum für Molekulare Neurobiologie and Department of Neurology, University Hospital Hamburg-Eppendorf, Martinistr. 52, D-20246, Hamburg, Germany

    Axel Methner

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  1. Jan Lewerenz
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  2. Susanne Thomsen
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  3. Julius A. Steinbeck
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  4. Axel Methner
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Correspondence to Axel Methner.

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Lewerenz, J., Thomsen, S., Steinbeck, J.A. et al. Short-term serum supplementation improves glucose-oxygen deprivation in primary cortical cultures grown under serum-free conditions. Methods Cell Sci 25, 227–236 (2004). https://doi.org/10.1007/s11022-004-9121-9

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  • DOI: https://doi.org/10.1007/s11022-004-9121-9

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