By Jeffrey W. Pollard, John M. Walker
Now thoroughly revised and up-to-date from the unique, much-acclaimed and bestselling first variation, simple mobilephone tradition Protocols, moment version deals modern such a lot accomplished selection of easy-to-follow, state of the art protocols for the tradition of quite a lot of animal cells. Its authoritative members supply particular, step by step directions, in addition to vast notes and counsel that let either specialists and novices to effectively in attaining their wanted effects. subject matters diversity from simple tradition technique to ideas for culturing formerly uncultured mobile forms and hard-to-culture differentiated cells. tools also are supplied for the research of residing cells via FACS, video microscopy, and confocal microscopy. just like the first version, this publication could be in each phone tradition laboratory and be of use to all who use mobilephone cultures in study.
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Extra info for Basic Cell Culture Protocols
To start the process, one should establish on tissue-culture plastic and m a serum supplemented medium (SSM) the minimal cell densities at which one can detect viable colonies (typically 100-I 000 tells/60-mm dish) when developing HDM for cell growth or the mmlmal cell densities for cell survival (typltally lO,OOO-100,000/60 mm) when developing HDM for optimal cell Ex Vivo Maintenance of Mammalian Cells 49 differentration. Use the lowest cell density at which one can routmely obtain colonies (m defimng growth condmons) or survival (for defimng dtfferentiation condittons) m SSM and on ttssue-culture plastic Then establish a negative control at the same density m serum-free medium (SFM): 1 Negative control cells on tissue-culture plastic and m SFM.
In particular, there is an update on some of the generic rules for defined ex vlvo conditions for cells (Tables 5-8). 1. Hormonal/y Defined Media (HDM) Containmg Rigorously Defined Nutrients, Hormones, and Growth Factors Any cell type can be maintained ex vivo, if it 1sprovided with appropriate substratum of extracellular matrix and with a serum-free HDM containing purified hormones, growth factors, trace elements, and nutritional factors (81,82). The exact composition of HDM 1stissue-specific and, very rarely, is species-specific.
1992) Use of fetal intestinal lsografis from normal and transgemc mice to study the programmmg of posltlonal mformatlon along the duodenal-to-colomc axis J Bzol Chem 267, 15,122-15,133 6 Henston, M. , Green, R. , and Gordon, J I. (1993) Chlmenc-transgenic mice represent a powerful tool for studying how the prohferatlon and dlfferentlatlon programs of intestinal eplthehal cell lineages are regulated. Proc Natl. Acad Scz USA 90,8866-8870 7 Huang, H and Auerbach, R. (1993) Identlficatlon and characterlzatlon of hematopoletic stem cells from the yolk sac of the early mouse embryo Proc Nat1 Acad Scz USA 90, 10,l l&10,1 14.