Oxidative Stress Protection in Cultured Rat Neurons

Cortical rat neurons were cultured as described 18 (link) from E21 rats or E17 mouse pups in Neurobasal-A medium and B27 (Invitrogen). Stimulations and transfections (Lipofectamine 2000, Invitrogen) were done at DIV8-10 after transferring neurons into trophically-deprived medium 18 (link). Action potential bursting was induced by treatment with 50 μM bicuculline, plus 250 μM 4-aminopyridine (Sigma) to enhance burst frequency. Stimulations were initiated 12 h prior to the application of an oxidative insult. Neurons were fixed after a further 24 h and subjected to DAPI staining and cell death quantified by counting (blind) the number of apoptotic nuclei as a percentage of the total. For details of analysis of peroxide-induced ROS accumulation and caspase activity, see supplemental methods.

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Papadia S., Soriano F.X., Léveillé F., Martel M.A., Dakin K.A., Hansen H.H., Kaindl A., Sifringer M., Fowler J., Stefovska V., Mckenzie G., Craigon M., Corriveau R., Ghazal P., Horsburgh K., Yankner B.A., Wyllie D.J., Ikonomidou C, & Hardingham G.E. (2008). Synaptic NMDA receptor activity boosts intrinsic antioxidant defences. Nature neuroscience, 11(4), 476-487.

Publication 2008

Action potential Aminopyridine Apoptotic Bicuculline Blind Caspase Cell death Cortical Dapi Lipofectamine 2000 Mouse Neurons Nuclei Peroxide Rats Transfections

Corresponding Organization :

Other organizations : University of Edinburgh, Harvard University, TU Dresden, Charité - Universitätsmedizin Berlin, Hutchison/MRC Research Centre, MRC Cancer Unit, Coriell Institute For Medical Research

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Variable analysis

independent variables

Stimulations and transfections (Lipofectamine 2000, Invitrogen)
Treatment with 50 μM bicuculline, plus 250 μM 4-aminopyridine (Sigma) to enhance burst frequency
Oxidative insult

dependent variables

Action potential bursting
Apoptotic nuclei as a percentage of the total
Peroxide-induced ROS accumulation
Caspase activity

control variables

Cortical rat neurons cultured as described 18 (link) from E21 rats or E17 mouse pups in Neurobasal-A medium and B27 (Invitrogen)
Neurons transferred into trophically-deprived medium 18 (link)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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