Download Animal Cell Culture Techniques by Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. PDF

By Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)

Cell tradition concepts enable numerous molecular and mobile organic inquiries to be addressed, delivering physiological stipulations when keeping off using laboratory animals. as well as easy ideas, quite a lot of specialized functional protocols overlaying the subsequent components are integrated: mobile proliferation and loss of life, in-vitro types for mobilephone differentiation, in-vitro versions for toxicology and pharmacology, commercial software of animal phone tradition, genetic manipulation and research of human and animal cells in culture.

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1985) such as changes in cell metabolism, alterations in cell karyotype and retardation of cell growth (Boyle et al. 1981; Sasaki et al. 1984; McGarrity et al. 1984). Mycoplasma are parasites and many are pathogens of a variety of animal hosts. ie Mary Heenan, Martin Clynes, National Cell and Tissue Culture Centre/BioResearch Ireland, Dublin City University, Glasnevin, Dublin 9, Ireland 3 Screening for Mycoplasma Contamination in Animal Cell Cultures 39 ever several human pathogenic mycoplasma have also been identified, including M.

Prepare overlay medium by combining 2x MM and agar. 3. Equilibrate in a 50 DC water bath and ensure no clumping occurs. 4. Prepare 10-fold (log) serial dilutions of virus suspension in MM diluent (10- 1-10- 8 ). plaque assay 34 EDWIN O'KELLY Fig. 11. Plaques of parainfluenza type 3 in VERO cells stained with 2 %(v/v) methylene blue (x20) 5. Remove the GM from the wells of the cluster plate. 6. 2 ml virus suspension (two wells for each dilution) and adsorb for 1 h at 37°C. Tilt the plate at IS-min intervals to prevent the monolayer from drying out and to ensure an even distribution of inoculum.

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.

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