Abstract
Primary neurons are difficult to cultivate because they are often part of a complex tissue, and synaptically connected to numerous other cell types. These circumstances often prevent us from unveiling molecular and metabolic mechanisms of distinct cells, as functional signals or assays cannot clearly be correlated with them due to interfering signals from other parts of the culture. We therefore present an up-to-date method for obtaining a highly purified neuronal culture of Purkinje cells. In the past, Purkinje cells were successfully isolated from young mouse cerebella, but this protocol was never adapted to other mammals. We therefore provide an updated and adjusted protocol for Purkinje cell isolation from rat instead of mouse cerebella. To purify Purkinje cells, we obtained perinatal rat cerebella, dissociated them and performed a Percoll gradient centrifugation to segregate the smaller and larger cell fractions. In a second step, we performed an immunopanning procedure to enrich only Purkinje cells from the large cell fraction. Based on former protocols, we used a different antibody for the immunopanning procedure and adjusted several aspects from the initial protocol to improve the yield and vitality of Purkinje cells. We provide RT-qPCR-based purity data obtained with this protocol and show the behaviour and the growth of these purified Purkinje cells. We provide a highly reproducible purification protocol for Purkinje cell cultures of high purity that allows functional analysis and downstream assays on living rat Purkinje cells and further morphological growth analysis in future.
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Abbreviations
- PC(s):
-
Purkinje cell(s)
- eGCL:
-
External granule cell layer
- iGCL:
-
Internal granule cell layer
- PCL:
-
Purkinje cell layer
- LMD:
-
Lasermicrodissection
- RT-qPCR:
-
Real-time quantitative PCR
- SD:
-
Standard deviation
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Acknowledgements
The authors gratefully thank FoRUM (RUB) for their financial support (F812-2014). The authors wish to thank C. Grzelak, A. Lodwig and S. Wenderdel for excellent technical assistance as well as A. Lenz for secretarial work. We would also like to give thanks to Daniel Terheyden-Keighley for helpful critical discussions.
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VT and CT designed the study. JT, LP and FW performed the experiments. JT and LP managed the literature searches and the statistical analysis. All authors wrote the manuscript in equal measure and approved the final manuscript.
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Tjaden, J., Pieczora, L., Wach, F. et al. Cultivation of Purified Primary Purkinje Cells from Rat Cerebella. Cell Mol Neurobiol 38, 1399–1412 (2018). https://doi.org/10.1007/s10571-018-0606-5
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DOI: https://doi.org/10.1007/s10571-018-0606-5