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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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
P19 cultures are started from a frozen stock by seeding cells into a T25 flask with
5 ml of P190M and allowing cells to reach nearconfluency. At this point, the
cultures have 5 to 8 x 10° cells total. Cells are passaged so that one-tenth of the
Then 0.5 ml of this cell suspension is used to inoculate each new flask that
contains 5 ml of P190M. Cells are passaged routinely every 2 days. Two aspects
of this protocol bear special attention. At low densities, embryonal carcinoma
To the undissociated tissue remaining in the flask, add additional HBSS and
enzyme solutions and incubate for 15min more. Remove the supernatant, add it
to the first supernatant, and filter the combined supernatants through a 72-pum
Transfer to tissue-culture flasks containing D-MEM supplemented with 10% FCS.
Use a density of about 2 x 10° cells per 25-cm” flask. This usually works out to 1
brain per 25-cm” flask or 2 to 3 per 75-cm” flask. The volume of medium used is 5
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