By Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)
Cell tradition options let quite a few molecular and mobile organic inquiries to be addressed, providing physiological stipulations while heading off using laboratory animals. as well as uncomplicated ideas, quite a lot of specialized functional protocols overlaying the subsequent parts are integrated: telephone proliferation and loss of life, in-vitro versions for mobile differentiation, in-vitro types for toxicology and pharmacology, commercial program of animal mobilephone tradition, genetic manipulation and research of human and animal cells in culture.
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Extra info for Animal Cell Culture Techniques
Weigh out 1 mg of powder and dissolve in 20 ml of sterile PBS by stir- ring on a magnetic stirrer for 30 min. 2. tm filter. 3. 0 ml aliquots, cover in aluminium foil and store in the dark at - 20°C. 4. A fresh stock should be prepared every 6 months, so an expiration date should be written on the aliquots. 5. tg/ml stock solution and diluting it in sterile PBS to 50 ng/ ml. Cover with aluminum foil and use immediately. Mounting Medium 1. 2 M disodium phosphate. 2. Mix with 50 ml of glycerol. 3.
Fig. 3. Stationary rack of inoculated tubes 2 Cell Culture and Diagnostic Virology 23 Standard Virus Isolation 1. Seed 1 ml cell suspensions at a concentration of approx. lOs cells/ml into standard culture tubes using freshly made GM. For viral isolation it is usual to prepare at least three different cell types for inoculation to increase the chances of isolation (Leland and French 1988). While some cell lines have a broad range of viral susceptibility, no single cell line is sensitive to every virus.
Hoechst 33258 stained NRK indicator cells, used in the analysis of a mycoplasma-free sample (a) and a sample contaminated with Mycoplasma hyo- rhinis (b) 46 MARY HEENAN, MARTIN CLYNES 11. Examine samples by flourescent microscopy for mycoplasma con- tamination. Optimum fluorescence excitation of the Hoechst 33258 stain occurs at approximately 360 nm, whereas emission peaks at approximately 475 nm. Therefore, excitation is performed with illumination from a high-pressure mercury lamp. Emission is best viewed through filter combinations that exclude light below 460 nm.