Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth.

Frontiers in Molecular Neuroscience
Julianne McCallArmin Blesch

Abstract

The development of eukaryotic transfection technologies has been rapid in recent years, providing the opportunity to better analyze cell-autonomous mechanisms influencing various cellular processes, including cell-intrinsic regulators of regenerative neurite growth and survival. Electroporation is one of the more effective methodologies for transfection of post-mitotic neurons demonstrating sufficient neuronal survival and transfection efficiency. To further maximize the number of transfected neurons especially with large plasmids, to limit the cellular exposure to serum, and to minimize the number of animals required for cell isolation per experiment, we compared two state-of-the-art electroporation devices for in vitro transfection of adult rat dorsal root ganglion (DRG) neuron cultures. By refining different parameters, transfection efficiencies of 39-42% could be achieved using the Lonza 4D-Nucleofector X-unit system, 1.5-2-fold higher rates than those that have been previously published for adult DRG neurons using smaller plasmid sizes. Our protocol further limits the number of cells required to 3 × 10(5) cells per 20 μl reaction using only 2 μg DNA/reaction and allows for the complete omission of serum post-transfection. ...Continue Reading

Citations

Oct 27, 2012·Annals of Biomedical Engineering·Adam J MellottMichael S Detamore
Feb 6, 2013·PloS One·Barbara ZavanPaolo Pinton
Feb 14, 2015·Cell·Christina HanackJan Siemens
Feb 24, 2015·Frontiers in Molecular Neuroscience·Lingli YuBrian B Rudkin
May 26, 2018·Science Translational Medicine·Gunnar H D PoplawskiMark H Tuszynski

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Methods Mentioned

BETA
transfection
light microscopy
transfections

Software Mentioned

Invitrogen
Neon
NeuronJ
Olympus Cell - P Software
NIH
ImageJ

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