Microinjection of SCG Neurons

Microinjection was performed as previously described^{16 (link)} with minor modifications. Dissociated cells were microinjected using a Zeiss Axiovert S100 microscope (Cambridge, UK) with an Eppendorf FemtoJet transjector and 5171 micromanipulator system and Eppendorf Femtotips (Stevenage, UK). Plasmids were diluted in 0.5 × PBS to a concentration of 100 ng/μl and passed through a Spin-X filter (Costar, Fisher, Loughborough, UK). The mix was injected directly into the nuclei of SCG neurons in dissociated cultures. All plasmids were co-injected with DsRed2 at a concentration of 25 ng/μl. In all, 70–200 neurons were injected per dish. Injection of relatively few neurons per dish facilitated visualization of individual labelled neurites, as neurites tend to cluster together in bundles.

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Di Stefano M., Nascimento-Ferreira I., Orsomando G., Mori V., Gilley J., Brown R., Janeckova L., Vargas M.E., Worrell L.A., Loreto A., Tickle J., Patrick J., Webster J.R., Marangoni M., Carpi F.M., Pucciarelli S., Rossi F., Meng W., Sagasti A., Ribchester R.R., Magni G., Coleman M.P, & Conforti L. (2014). A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration. Cell Death and Differentiation, 22(5), 731-742.

Publication 2014

Axiovert S100 microscope Femtotips Spin-X filter

Cells Dish Microinjection Microscope Neurites Neurons Nuclei Plasmids S100

Corresponding Organization :

Other organizations : Queen's Medical Centre, University of Nottingham, Babraham Institute, Marche Polytechnic University, University of Edinburgh, University of California, Los Angeles, Doheny Eye Institute, Università di Camerino

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Variable analysis

independent variables

Microinjection method (Zeiss Axiovert S100 microscope, Eppendorf FemtoJet transjector and 5171 micromanipulator system, and Eppendorf Femtotips)
Plasmid concentration (100 ng/μl)
Co-injection of DsRed2 (25 ng/μl)

dependent variables

Visualization of individual labelled neurites

control variables

Dissociated SCG neurons in culture
Injection of 70-200 neurons per dish
Spin-X filter to pass the plasmid mix

controls

Positive control: DsRed2 co-injection to visualize injected neurons

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