By Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)
Cell tradition concepts permit numerous molecular and cellphone organic inquiries to be addressed, providing physiological stipulations when heading off using laboratory animals. as well as uncomplicated ideas, a variety of specialized functional protocols overlaying the next parts are integrated: phone proliferation and loss of life, in-vitro versions for mobilephone differentiation, in-vitro versions for toxicology and pharmacology, commercial software of animal mobilephone tradition, genetic manipulation and research of human and animal cells in culture.
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Additional info for Animal Cell Culture Techniques
Materials NRK cells in log phase (ATCC; CRL 1570) 35 mm petri dishes Microscope slides Coverslips Sterile PBS Hoechst 33258 Mounting medium - Mycoplasma orale (ATCC; 45539) 42 preparation of solutions and equipment MARY HEENAN, MARTIN CLYNES Mycoplasma hyorhinis (ATCC; 23234) Carnoy's fixative Carnoy's/PBS Fluorescent microscope Hoechst 33258 Stock Note: This stain is carcinogenic. Care should be taken when handling the stain, gloves should be worn and waste must be disposed of according to local regulations.
2. Mix the ingredients at 37°C until dissolved. Bring the final volume up to 250 ml with distilled water. 3. -lm filter. 4. Dispense into 25 ml aliquots and store at -70°C until required. Mycoplasma Broth Medium 1. 005 g phenol red to 150 ml distilled water. Heat to dissolve. 2. Sterilize the solution by autoclaving. 3. Allow the broth mixture to cool to room temperature. 4. Aseptically add 50 ml serum, 25 ml yeast extract (15 % w/v) and 25 ml stock solution. Mix the solution completely. 4. 5. Dispense 5 ml aliquots of the broth medium into sterile test tubes and store at 4°C, Use within 3 -4 weeks.
5 % sodium bicarbonate. 2. Disperse confluent monolayers of cells by washing twice with equal volumes of a preheated versene/trypsin mixture. 3. 4. Scheme for dividing plate into four sections Rows Rows Rows Rows A-D A-D E-H E-H Columns Columns Columns Columns 1-6 7 -12 1-6 7 -12 3. 3). 4. Divide the plate (Fig. 4. 5. Add 90 ~l serum-free medium to each well of a V-bottomed cell culture microtiter plate. 6. Add 10 ~l of the first specimen to row A wells 1-6. 7. Similarly add 10 ~l of the second, third and fourth specimens to row A wells 7 -12, row E wells 1-6, and wells 7 -12, respectively.