By Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)
Cell tradition suggestions enable a number of molecular and mobile organic inquiries to be addressed, delivering physiological stipulations while keeping off using laboratory animals. as well as easy thoughts, a variety of specialized useful protocols overlaying the subsequent parts are integrated: mobilephone proliferation and dying, in-vitro types for phone differentiation, in-vitro versions for toxicology and pharmacology, commercial software of animal mobile tradition, genetic manipulation and research of human and animal cells in culture.
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Extra info for Animal Cell Culture Techniques
Seed 2 ml of cell suspension containing 1 x 105 cells per ml into the wells of a 24-well cell cluster plate and allow to form a healthy, confluent monolayer (2-3 days). 2. 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.
3 Screening for Mycoplasma Contamination in Animal Cell Cultures 49 5. Proceed immediately to dispense 10 ml aliquots into 60 mm petri dishes. Add the fluid as quickly as possible in order to eliminate the problem of the agar solidifying before the medium is completely dispensed. The plates may be stored at 4°C for up to 4 weeks . • Procedure 1. Samples, including positive and negative controls, should be set up in duplicate. Agar plates should be labeled on both the top and base, and it should be possible to differentiate between replica plates.
Some viruses (influenza, parainfluenza) need to be cultured at 33 °C with trypsin but without fetal calf serum in the MM. 7. Examine the cultures daily for CPE. Microtiter Method of Virus Isolation This method represents an enhancement of conventional monolayer isolation techniques (Fong and Landry 1991; Brumback et al. 1995). Using this method six cell lines are seeded in suspension on microtiter plates thereby improving the sensitivity of virus isolation (O'Neill et al. 1996). Up to four specimens can be inoculated with each plate.
Animal Cell Culture Techniques by Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)