By Melany Jackson, A. Helen Taylor, Elizabeth A. Jones (auth.), Andrew Ward, David Tosh (eds.)
Cultured cells have mixed accessibility and the facility to extend a homogeneous mobile inhabitants from a comparatively constrained resource, hence establishing up a wealth of percentages for researchers. In Mouse cellphone tradition: equipment and Protocols, professional researchers supply a couple of tools for the tradition of a variety of particular cells and tissues remoted from the major genetic version of the fetal or grownup mouse. together with protocols for the explant of fetal tissues and stem cells that let developmental procedures to be ex vivo in addition to protocols for the tradition of remoted telephone kinds that let for the learn of rather homogeneous mobilephone populations, this quantity brings jointly a variety of the most up-tp-date equipment so as to cause them to to be had in a single handy resource. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, easily reproducible laboratory protocols, and notes on troubleshooting and fending off identified pitfalls.
Practical and authoritative, Mouse cellphone tradition: equipment and Protocols serves as an instantly appropriate springboard for the improvement of recent tissue tradition tools in an effort to develop the research and remedy of human issues.
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Extra resources for Mouse Cell Culture: Methods and Protocols
8. Lab centrifuge (Heraeus Labofuge 300; Kendro Laboratory Products, Germany). 9. Ice bucket and ice. 10. Pasteur pipettes. 11. Cells cultured to exponential phase (∼75% confluent). 34 Smith and Merrick 12. Freshly made fixative (3:1 v/v methanol: glacial acetic acid). 13. Coverslips and DePEX (Merck) mounting fluid. 14. Upright microscope (Nikon Eclipse E600; Nikon, UK). 3. Establishing Primary Microexplant Cultures from Embryos 1. Pregnant mice at the appropriate stage of gestation. 2. Sterile dissection instruments.
Dimethylsulphoxide (DMSO). 5. Freeze down mix: 10% DMSO in DF10 medium. 6. –80◦ C Freezer, liquid nitrogen storage tank. 3. Determining Cell Numbers 1. A Neubauer haematocytometer. 2. Haematocytometer coverslips. 3. Inverted microscope (Leica DMIRB, Leica, UK). Embryonic Skeletal Muscle 33 4. Hand counter. 5. Pipettes and hand-held pipetting device. 2. Establishing Primary Skeletal Muscle Microexplant Cultures 1. Freshly dissected skeletal muscle from aged (16 months or older), adult (2–15 months) or juvenile (1–8 weeks) mice.
Following centrifugation the supernatant is removed and the cells are washed by re-suspending the cell pellet in a further 5 ml DF10, then centrifuge as before. 3. The cell pellet is then mixed for the second time with 5 ml of DF10 and the resulting cell suspension is transferred to a small 25 cm2 plastic culture vessel. 4. Cultures are maintained at 37◦ C in a humidified incubator containing 5% CO2 in air. Unless vessels are used with a filtered cap, the cap of the flask must be slightly loosened for 38 Smith and Merrick several hours to allow air in the culture vessel to equilibrate with the incubator and acidify the culture medium.