Thaw a vial of primary precursor osteoclasts in a 37°C water bath. 2. After thawing, transfer the cells to a 15 mL centrifuge tube, add 10 mL of Wash Medium and mix briefly. Centrifuge 1,000 rpm for 5 minutes at 4°C. 3. Remove supernatant and add 10 mL of Wash Medium and mix briefly. Centrifuge 1,000 rpm for 5 minutes at 4°C. 4. Remove supernatant and resuspend the cells in 2.5 5 mL of Culture Medium. To study factors that effect osteoclasts formation, add the factors to the Culture Medium. 5. Transfer 100 µL of cell suspension into each well of a 96-well plate. If the cells are resuspended in 5 mL of Culture Medium, there will be enough cell suspension for about 50 µLs. To quickly observe osteoclasts formation, culture the cells at a higher density. 6. Feed the cells with 100 µL of Culture Medium every 3 4 days. Cells will begin to fuse and form osteoclasts around day 5 (fig 1). 7. Count the osteoclasts by staining with tartrate-resistant acid phosphatase (TRAP Staining Kit,.
限制
仅限研究用
注意事项
1. Read the instructions carefully before beginning the culture. 2. This kit is for research use only, not for human or diagnostic use.
抗原
Osteoclast
背景
In aging societies, osteoporosis and other age-related bone metabolism disorders are a rapidly increasing problem. The amount of bone in an organism is controlled by a balance of osteoblasts (bone-forming cell) and osteoclasts (bone- destroying cell) activities. A method to induce osteoclasts formation from bone marrow cells using M-CSF (macrophage- colony stimulating factor) and RANKL (receptor activator of NF-B ligand) has been established in recent years. This kit includes cryopreserved primary precursor osteoclasts from rat bone marrow and Culture Medium containing M-CSF and RANKL.