-
- Target
- Osteoclast
- Reactivity
- Rat
- Application
- Cell Culture (CC)
- Components
-
2x Rat Osteoclast Precursor Cells, frozen. Vial containing 2 x 10^6 cells
1x Washing Medium, alpha-MEM. 50 mL
1x Culture Medium, alpha-MEM with M-CSF (10 ng/mL) and RANK Ligand (10 ng/mL). 25 mL - Material not included
-
Pipettes
96-well, flat bottom culture plate
Tubes
Refrigerated centrifuge
Water bath.
-
- Calculation of Results
-
- 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,.
- Thaw a vial of primary precursor osteoclasts in a 37°C water bath.
- Restrictions
- For Research Use only
-
- Handling Advice
-
1. Read the instructions carefully before beginning the culture.
2. This kit is for research use only, not for human or diagnostic use.
-
- Target
- Osteoclast
- Background
- 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.
-
You are here:
