Matrigel invasion assays
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2035
| Overview | |
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Matirgel is considered as basement membrane and generated from EHS sarcoma. Matrigel contains not only basement membrane components (collagens, laminin, and proteoglycans)but also matrix degrading enzymes/their inhibitors and growth factors. Invasion of tumor cells into Matrigel has been used to characterize involvement of ECM receptors and matrix degrading enzymes which play roles in tumor progression. |
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| Material | |
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- Matrigel (Becton-dickinson) - 24-transwell (Coster) - Fibronectin(Sigma) - Diff-Quick staining solution (Fischer Scientific) |
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| Procedure | |
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1. Thaw Matrigel at 4C overnight. 2. Dilute Matrigel (5mg/ml to 1 mg/ml) in serum free-cold cell culture media (RPMI1640, EMEM, DMEM, etc). 3. Put 100 ul of the diluted matrigel into upper chamber of 24-well transwell 4. Incubate the transwell at 37C at least 4 to 5 h for gelling. 5. Harvest cells from tissue culture flasks by Trypsin/EDTA. 6. Wash the cells 3 times with culture media (RPMI1640, EMEM, DMEM etc)containing 1 % FBS. 7. Resuspend the cells in media containing 1% FBS at a density of 10^6 cells/ml. 8. Gently wash gelled matrigel with warmed serum free-culture media. 9. Put 100 ul of the cell suspension onto the matrigel. 10. lower chamber of the transwell is filled with 600 ul of culture media containing 5 ug/ml fibronectin, as an adhesive subtrate. 11. Incubate at 37C for 20 to 24 h. 12. Remove transwells from 24-well plates and stained with Diff-Quick solution. 13. Scrape off noninvaded cells on the top of the transwell with a cotton swab. 14. Count invaded cells under a light microscope. |
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| Troubleshooting | |
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- Need to check batch of matrigel. - Matrigel tends to form gel very quickly at room temerature, therefore, pipets and tips using in steps 2 and 3 have to be chilled at -20C prior to experiements. - In our experience, matrigel would not make gel under a concentration of 1 mg/ml. - If cell make aggregation during invasion assays, reduce the density of cell suspension (at step 7). - Invasion assays can be performed for 36 to 40h. |
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| Reference | |
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Knutson, JR., Iida, J., Fields, GB, and McCarthy, JB. Molecular Biology of the Cell, 7: 383-396, 1996. |
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