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