By Miep H. Helfrich, Stuart H. Ralston
The decade has visible an incredible improve in our figuring out of bone biology. The genes answerable for the vast majority of infrequent inherited bone issues were pointed out and lots more and plenty growth has been made within the id of genes in polygenic problems resembling Paget’s ailment and complicated multigene ailments similar to osteoporosis. Transgenic expertise has pointed out additional genes, occasionally suddenly, with profound results on bone. This wealth of latest genetic info will surely result in huge mobile organic experiences to appreciate the mechanisms through which those gene items have an effect on bone mass and bone energy. In Bone learn Protocols a list of protocols has been assembled to accomplish such mechanistic experiences. within the culture of the equipment in Molecular drugs sequence, the chapters are useful laboratory protocols that are meant to allow the reader to hold out the options from scratch. To our wisdom this is often the 1st time this sort of really useful guide on well-established bone equipment has been assembled, and this quantity goals to be complementary to and keep on with on from the extra theoretical equipment in Bone Biology, edited through Arnett and Henderson (1).
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10 and 11), scissors, tweezers, and curved forceps. 3. 2-µm disposable filter units. 02/Nulend/19-28/F1 20 2/26/03, 10:44 AM Bone Cells from Calvariae and Long Bones 21 4. 25-cm2 tissue culture flasks (Nunc), six-well tissue plates (Costar), 94/16-mm cellstar Petri dishes (Greiner), and 145/20-mm cellstar (large) Petri dishes (Greiner). 5. , Staffordshire, UK). 3. Media and Solutions 1. 1 mM Na2HPO4. 4. 2. 4. 3. 25 µg/mL of fungizone (Gibco), 100 µg/mL of ascorbate, and 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA; see Note 1).
Wash in HBSS and add wash to fraction 4. 8. Further fractions can be collected by repeating steps 5 and 6, but cell yields will be increasingly lower. 9. ). 10. Plate out pooled or single fractions in 75-cm2 culture flasks using cells derived from two or three animals per flask. Later fractions will contain the most differentiated cells. Cells isolated in this way (pooled fractions 2–4) have been used successfully in cocultures to generate osteoclasts (see Chapter 11 by van ’t Hof, this volume).
4. Kodak DC50 digital camera or similar. Good quality white light transilluminator. Adobe Photoshop or similar image editing software. Intelligent quantifier or similar colony counting software. 3. 1. Isolation of Whole Bone Marrow Cells 1. Use four 125–200-g male Wistar rats and euthanize these by cervical dislocation (see Note 4). 03/Scutt/29-40/F1 31 2/26/03, 10:44 AM 32 Scutt et al. Fig. 2. Recovery of tibial and femoral bone marrow cells using a centrifugation method. Bone marrow cells are isolated using a brief centrifugation step.