Culturing Nerve Cells
A do-it-yourself manual for culturing nerve cells, complete with recipes and protocols.
Because neurons and glia in culture are remarkably similar to those in situ, culture systems make it possible to identify significant cell interactions and to elucidate their mechanisms. This book is in many ways a do-it-yourself manual for culturing nerve cells, complete with recipes and protocols. But it also provides an understanding of the principles behind the protocols. In effect the contributors invite you into their labs and provide much of the information you would obtain from such a visit.The authors of the introductory chapters present the nuts-and-bolts principles of growing nerve cells. The authors of the following chapters discuss the culturing of specific cell types. They explain how their experimental goals have shaped their particular cell culture approach and the advantages and disadvantages of the cell culture systems they have developed. They provide detailed protocols and describe their cultures in practical terms, from when the cells are first plated through the various phases of their development.
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These methods make use of the natural means viruses have evolved for ensuring
that their DNA reaches the nucleus of infected neurons, while in most cases
debilitating the virus so it will not undergo a full lytic cycle. The most commonly
Then, the amplicon is transfected into a packaging cell line by standard methods,
such as lipid-mediated transfection. Finally, the transfected cells are
superinfected with helper virus to provide HSV replication and packaging
functions in trans.
A pBR322 amp pNF314-P1-lacz-pA HSV-1 packaging site B E5 cells 99. F. Lipid-
mediat pNF-lacz transfection -> Co-infection HSV d120 IE3 deletion mutant
helper virus HSV-1 ! On () + E34. } Defective HSV Helper expressing lacz virus C
Remove the virus-containing medium from the flask and add 4 ml of fresh
medium containing 8 x 10° E5 cells obtained by trypsinizing the extra flask of E5
cells seeded on day 1. The purpose of overlaying cells with each passage is to
P3 or P4 stocks usually are optimum for neuron transfection, with titers of 10°-10°/
ml (before concentration) and approximately equal ratios of packaged amplicon
to helper virus. Further passaging tends to decrease titers without improving the ...
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