By Jeffrey W. Pollard, John M. Walker
Animal cellphone tradition, the newest quantity in Humana's hugely winning tools in Molecular Biology sequence, presents distinctive sensible options for the tradition of a huge spectrum of simple cellphone phone kinds. Chapters supply hands-on equipment for growing mammalian fibroblastic mobile cultures and conserving tradition stipulations for epithelial, neuronal, and hematopoietic cells between others. consciousness is given to the variety of tradition media and extracellular matrices had to retain the differentiated services of the aesthetic cells. The book's particular energy lies in its descriptions of tradition suggestions for either dwelling and stuck cells. Chapters conceal concepts resembling: • cinematographic research • in situ mRNA hybridization • immunofluorescence • immunoelectron microscopy • somatic telephone hybridization • DNA transformation • insect cellphone tradition • construction of hybridoma cellphone traces • monoclonal antibody concepts • new, really good methodologies. an invaluable appendix lists the main common tradition media. complete in scope and assurance, and punctiliously up to date, Pollard and Walker's specified guide on ANIMAL cellphone tradition is an critical source for either the amateur and the pro specialist.
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Extra info for Animal Cell Culture (Methods in Molecular Biology Vol 5)
4. EBV suspension, 5 x lo4 Transforming Units (TFU). 5. F(ab’), fragment of goat anti-human IgM (p-chain specific). 6. Ficoll-paque. 7. RPM1 growth medium contains 5,10, or 15% fetal calf serum, 2 mM Lglutamine, 100 pg/mL penicillin, and 100 mg/mL streptomycin. 8. Growth medium containing 10% FCS, and 2-Mercaptoethanol (2 x lo4 M). 9. Hanks’ balanced salt solution pH 4 (seeAppendix). 10. Helix pomatia Lectin-Sepharose 6MB. 11. Heparin. 12. IgG: antihuman. 13. IgG: Pan T or Pan B. 14. Interleukin 2,10-40 U/mL final cont.
11. At this point, cells can be set up immediately for transformation or stored in liquid nitrogen (seeNote 3). 3. Cell-Freezing Procedure 1. Centrifuge the cell suspension at 1508 for 4 min. 2. Resuspend the pellet in the correct vol of 91% newborn calf serum (NBCS) + 9% DMSO to achieve 5 x lo6 cells mL. 3. Freeze in a programmable freezer at -3’C/min. Alternatively, freeze overnight in a polystyrene container in a -8OOCrefrigerator and then remove ampules to -196OC. 4. Virus Transformation Either: 1.
The scheme is shown diagrammatically in Fig. 3. Many fiber cartridges are not steam sterilizable, but polysulfone and teflon fibers are. The quantity of medium withdrawn must be balanced by adding fresh medium from a reservoir. Using flow rates of up to 1 vol/h, cell densities in the region of 2-5 x 107/mL can be achieved, but oxygen is a limiting factor and an additional filtration cartridge should be put in the external loop as an oxygenator. Scale- Up of Animal Cells 55 machine press direction A Fig.
Animal Cell Culture (Methods in Molecular Biology Vol 5) by Jeffrey W. Pollard, John M. Walker