Methods for culturing primary sympathetic neurons and for determining neuronal viability.
Developing nerve growth factor (NGF)-dependent sympathetic neurons are one of the best-studied in vitro models of neuronal apoptosis and have been used to identify key components of the neuronal cell death pathway. This chapter describes how to prepare purified cultures of primary sympathetic neurons and how to induce apoptosis by NGF deprivation. In addition, a simple method for measuring neuronal viability based on the live/dead assay is also described. This can be used for assessing the effect of small molecule inhibitors of protein kinases, caspases and other enzymes, on NGF withdrawal-induced death.
Whitfield
Jonathan
J
Eisai London Research Laboratories, University College London, UK.
Neame
Stephen J
SJ
Ham
Jonathan
J
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001681:Biological Assay / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D048429:Cell Size; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D020932:Nerve Growth Factor / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D013564:Sympathetic Nervous System / Q000166:cytology*
D003470:Culture Media; D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; D020932:Nerve Growth Factor