By Simon P. Langdon (auth.), Simon P. Langdon (eds.)
The turning out to be of melanoma cells within the laboratory has been a strong software for learning and figuring out the biology of melanoma and the results of gear on melanoma cells. In melanoma mobile tradition: tools and Protocols, specialist researchers describe intimately their most efficient and updated equipment for growing to be melanoma cells within the laboratory. appropriate for beginner and skilled researchers alike, those comfortably reproducible innovations remedy a various variety of experimental difficulties. there are ways to represent and authenticate cellphone strains, to isolate and boost particular different types of melanoma cells, and to boost new cellphone line versions. useful assays are supplied to guage clonogenicity, cellphone proliferation, apoptosis, adhesion, migration, invasion, senescence, angiogenesis, and mobilephone cycle parameters. different tools let the amendment of melanoma cells for transfection, improvement of drug resistance, immortalization, and move in vivo; the coculture of other telephone forms; and the detection and therapy of illness. every one absolutely validated protocol is defined in step by step element via a longtime professional within the box and encompasses a historical past creation explaining the primary in the back of the method, apparatus and reagent lists, and pointers on troubleshooting and warding off identified pitfalls.
accomplished and hugely functional, melanoma telephone tradition: tools and Protocols offers either simple scientists and scientific researchers with a gold-standard number of complex strategies for culturing melanoma cells successfully of their laboratories.
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Extra info for Cancer Cell Culture: Methods and Protocols
1. Adherent Monolayer 1. For monolayer cultures that are not being subcultured, remove spent media with a sterile pipet and add an equal volume of fresh media (plus serum plus any additives). 2. The periodicity of feeding depends on the growth rate of the primary culture or cell line. In general, feeding 2–3 times/wk is recommended. 2. Suspension Culture 1. For suspension cultures, medium containing cells must be centrifuged in a sterile manner, at 600g for 5 min in a Universal container. 2. The cell pellet is resuspended in fresh medium.
Gently move the cover slip back and forth resulting in its attachment to the 26 Macleod and Langdon hemocytometer and the appearance of Newton’s rings (rainbow colors like those formed by oil on water). 5. The hemocytometer is now ready to be filled. Place a pipet filled with a well-suspended mix of cells at the edge of the coverslip and then by slowly expelling some contents, draw the fluid into the chamber by capillary action. 6. Obtain the cell concentration by counting cells in the grid area.
5X final density. 3. Cell culture media (for example, Ham’s F12 plus 10% FCS) is warmed to 37°C. 4. Using a pipet or pastette, add 2 mL of 5% agar to 18 mL cell culture medium in a prewarmed glass-Universal and mix thoroughly. 5. 4 mL of the cell suspension in individual tubes (with caps that allow diffusion) and mix thoroughly. 6. Place tubes on ice for 5 min and close caps. Then place tubes into a CO2 incubator at 37°C. 7. After 1 wk, 1 mL fresh medium plus serum can be added to each tube. The tube is re-fed weekly until colony size is greater than 50 cells.
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