By Miep H. Helfrich, Stuart H. Ralston
Experiences over the last decade have persevered to carry super advances to our knowing of bone biology. New pathways were found and multiplied our wisdom of the ways that genes and gene items have an effect on bone cells and thereby bone mass and bone power. In Bone examine Protocols, moment version, professional researchers within the box element many equipment conventional to check bone biology. Focusing typically on in vitro tools, this quantity offers options for isolation, tradition and sensible research of all bone mobile forms and information quite a number imaging equipment, together with mild and ultrastructural microscopy and stay mobilephone imaging. a few very important in vivo thoughts are integrated, comparable to research of bone resorption and imaging utilizing X rays, fluorescent or luminescent innovations. equipment for learn of proteins and nucleic acid are incorporated and techniques for research of bone composition, size of bone energy, and reaction to mechanical stimulation are defined. Written within the hugely profitable tools in Molecular Biology™ sequence structure, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and key tips about troubleshooting and keeping off recognized pitfalls. Authoritative and sensible, Bone examine Protocols, moment variation seeks to assist scientists within the bone box to set up new strategies of their laboratories.
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Extra resources for Bone Research Protocols (Methods in Molecular Biology, v816, 2nd Ed.)
1. Polystyrene plate and needles for fixing the mice. 2. Scalpels (no. 10 and 11), scissors, tweezers, and curved forceps. 3. 2 μm disposable filter units (Schleicher & Schuell GmbH, Dassel, Germany). 4. 25 and 75 cm2 tissue culture flasks (Nunc, Roskilde, Denmark), 94/16 mm cellstar petri dishes (Greiner), and 145/20 mm cellstar (large) petri dishes. 5. 100 × 16 mm (10 ml) conical base test tubes with screw cap (Bibby Sterilin ltd, Staffordshire, UK) and conical base, 15 and 25 ml polypropylene centrifuge tubes (Greiner).
4. Notes 1. Addition of serum to the medium is necessary for the survival and stimulation of proliferation of the primary mouse bone cells. However, “serum” is not a constant and homogeneous product, and the growth rate of primary bone cells can vary considerably between several batches of serum. It is therefore recommended to test several batches of serum on their cell proliferative ability and continue to use the one that produces the best results. 2. Sometimes, the primary bone cultures can contain fibroblasts, which grow faster than the bone cells and can quickly overgrow the primary bone cell cultures.
Chem. 276, 9565–9571. 19. Gundle, R. , Joyner, C. , and Triffitt, J. T. (1995) Human bone tissue formation in diffusion chamber culture in vivo by bone derived cells and marrow stromal cells. Bone 16, 597. 20. , Joyner, C. , and Triffitt, J. T. (1997) Interactions of human osteoprogenitors with porous ceramic following diffusion chamber implantation in a xenogeneic host. J. Mater. Sci. Mater. Med. 8, 519–523. 21. , Jordan, G. , and Beresford, J. N. (2000) Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1–4.
Bone Research Protocols (Methods in Molecular Biology, v816, 2nd Ed.) by Miep H. Helfrich, Stuart H. Ralston