Abstract
Despite international research efforts, patients with glioblastoma multiforme (GBM)—the most common malignant brain tumors in adults—exhibit a very unfavorable prognosis. Their aggressive local growth pattern and increased invasiveness, due to a high motility of the tumor cells, hamper treatment. However, the molecular mechanisms regulating glioblastoma cell migration are still elusive. Here, we describe the combination of a highly efficient cell transfection by Nucleofection® technology and the generation of spheroids from these transfected glioblastoma cell lines. Nucleofection allows the manipulation of protein expression by overexpression and siRNA mediated protein knockdown. Transfection efficiencies >70% can be achieved with some GBM cell lines. Transfected neurospheres then can be used for migration assays (as described here in detail) and a multitude of other functional assays. In comparison to monolayer cultures, the advantage of spheroids is their resemblance with organized tissue in combination with the accuracy of in vitro methodology and marked experimental flexibility.
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Acknowledgments
We are very grateful to Siglinde Kühnel and Elisabeth Karl for technical assistance. We also thank Alexandra Schock for the data she produced during an internship in the laboratory.
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Hagemann, C., Amend, D., Kessler, A.F., Linsenmann, T., Ernestus, RI., Löhr, M. (2017). High-Efficiency Transfection of Glioblastoma Cells and a Simple Spheroid Migration Assay. In: Zhang, B. (eds) RNAi and Small Regulatory RNAs in Stem Cells. Methods in Molecular Biology, vol 1622. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7108-4_5
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DOI: https://doi.org/10.1007/978-1-4939-7108-4_5
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