Matrigel Invasion Assay Protocol Overview of Protocol
Matrigel 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. Materials
- Matrigel (Becton-****inson)
- 24-transwell (Coster)
- Diff-Quick staining solution (Fischer Scientific) Protocol
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. Troubleshooting
- 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. References
Knutson, JR., Iida, J., Fields, GB, and McCarthy, JB.
Molecular Biology of the Cell, 7: 383-396, 1996.
Protocol by Joji Iida.