By Miep H. Helfrich, Stuart H. Ralston
Studies during the last decade have persisted to carry large advances to our knowing of bone biology. New pathways were stumbled on and accelerated our wisdom of the ways that genes and gene items impact bone cells and thereby bone mass and bone power. In Bone examine Protocols, moment Edition, specialist researchers within the box element many equipment regularly occurring to check bone biology. Focusing quite often on in vitro equipment, this quantity offers concepts for isolation, tradition and sensible research of all bone telephone forms and info quite a number imaging tools, together with gentle and ultrastructural microscopy and reside phone imaging. a few vital in vivo strategies are incorporated, equivalent to research of bone resorption and imaging utilizing X rays, fluorescent or luminescent options. equipment for learn of proteins and nucleic acid are incorporated and strategies for research of bone composition, size of bone energy, and reaction to mechanical stimulation are defined. Written within the hugely winning Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols, and key tips about troubleshooting and warding off recognized pitfalls.
Authoritative and sensible, Bone study Protocols, moment Edition seeks to help scientists within the bone box to set up new recommendations of their laboratories.
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Extra info for Bone Research Protocols (Methods in Molecular Biology)
Roberson, P. , and Manolagas S. C. (1998) Loss of estrogen upregulates osteoblastogenesis in the murine bone marrow. Evidence for autonomy from factors released during bone resorption. J. Clin. Invest. 101, 1942–1950. 20. , et al. (1997) Increased adipogenesis and myelopoiesis in the bone marrow of SAMP6, a murine model of defective osteoblastogenesis and low turnover osteopenia. J. Bone Miner. Res. 12, 1772–1779. 21. Dobson, K. , and Scutt, A. (1999) Centrifugal isolation of bone marrow from bone: an improved method for the recovery and quantitation of bone marrow osteoprogenitor cells from rat tibiae and femurae.
5 mL of the resultant suspension is used in the culture. In this way the number of CFU-f per bone can be calculated and is largely independent of changes in the overall bone marrow cell population. 7. The overall cell viability is between 70% and 80%. 8. Acquiring images for colony quantitation: Position the camera stand so that the Petri dish fills as much of the image as possible. With careful positioning, images from dishes that have been stained sequentially can be easily aligned. There are now a good selection of digital cameras suitable for this type of work.
Tive. Counting should ideally be performed “blind” to the treatment group. After counting osteoclasts, the numbers of mononuclear cells can be assessed using toluidine blue staining to ensure that cells were settled at similar densities onto the discs and that agents tested did not have a general cytotoxic effect. Cell counting should be performed in a “blinded” manner.