By Miep H. Helfrich, Stuart H. Ralston
Reports during the last decade have persisted to convey large advances to our realizing of bone biology. New pathways were stumbled on and elevated 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 version, professional researchers within the box element many equipment generic to check bone biology. Focusing frequently on in vitro equipment, this quantity offers suggestions for isolation, tradition and useful research of all bone cellphone kinds and information a number imaging tools, together with mild and ultrastructural microscopy and dwell cellphone imaging. a few vital in vivo concepts are incorporated, resembling research of bone resorption and imaging utilizing X rays, fluorescent or luminescent innovations. equipment for examine of proteins and nucleic acid are incorporated and strategies for research of bone composition, size of bone power, and reaction to mechanical stimulation are defined. Written within the hugely winning equipment in Molecular Biology™ sequence structure, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, quite simply reproducible laboratory protocols, and key tips about troubleshooting and warding off recognized pitfalls. Authoritative and sensible, Bone study Protocols, moment variation seeks to help scientists within the bone box to set up new suggestions of their laboratories.
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Extra resources for Bone Research Protocols (Methods in Molecular Biology, v816, 2nd Ed.)
The advantage of cell lines over freshly isolated cells lies in the immediate availability of large numbers of cells, the homogeneity of the cell cultures, and the expected stability of the phenotype. In the long run, however, many cell lines appear unstable to some extent, and subclones of cell lines tend to develop in different laboratories. In addition, the clonal selection generally favors rapidly growing cells, but these might not express all typical features of cells from a certain tissue, and the resulting cell line may thus not be entirely representative.
To form mineralised bone nodules in vitro, an additional source of phosphate is required. The most widely used phosphate source is β-GP, although phosphate itself can also be used. Figure 3 illustrates the critical importance of β-GP concentration in this culture system (see also ref. 6). In osteoblast cultures lacking β-GP, organic matrix is deposited but mineralisation fails to occur. Osteoblasts cultured with 2 mM β-GP reproducibly form abundant bony structures with characteristic “trabecular” morphology; alizarin red staining shows that mineralisation is confined to these matrix structures.
Add 700 μl FBS to the cell suspension to inhibit trypsin activity. 7. ), shake well, and add the supernatant to the tube containing the cell suspension. This is population number 1. 8. Add new digestion solution to the calvaria, and repeat the previous three steps to obtain population number 2. D. Bakker and J. Klein-Nulend centrifuge cell population number 1 at 300 × g for 5 min. Discard supernatant, resuspend cell pellet in 1 ml cCM, and add to 17 ml cCM. Pipette in a 6-well plate at 3 ml cell suspension per well.
Bone Research Protocols (Methods in Molecular Biology, v816, 2nd Ed.) by Miep H. Helfrich, Stuart H. Ralston