Matrigel Invasion Assay Protocol

[moleculardude]

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)
- Fibronectin(Sigma)
- 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.

[aawasthi]

Hi,

I was trying the invasion assay according to your protocol. I got matrigel from BD and it is at a conc of 8.5 mg/ml.BD suggested not to dilute the matrigel more than 1:3 so i was doing the assay at this dilution. The trouble I am having is that i see the migration of the cells only in the center of the insert and also when i remove the cells from the top of the membrane after 24hrs, the gell looks a bit liquidy and not a solid gel. Is it suppose to be solid or like this. And what would be the best way to pour 100 ul of the diluted matrigel onto the membrane so that it forms a uniform layer.

thanks for any suggestions

[ga260]

Hello,

I was wondering whether Matrigel is used for keratinocytes. It seems to be used for epithelial cells but I haven't seen an example with keratinocytes. Is there a specific reason? Has it been tried? I would be very happy if someone can help me in this case.

Thanks very much.

[guynextdoor84]

may i know is it one should use low concerntration of matrigel so that the cells can invade? higher concerntration would cause false negative result? does matrigel concerntation correlate with number of cells manage to invade?

[munchems]

Can the prepared plates be frozen and thawed at a later time? Would they need to be rehydrated (as the pre-prepared invasion plates from BD need to be) after thawing, but prior to use?

[q77]

Does anyone know what the purpose of fibronectin is in the matrigel invasion assay? Is it necessary to use it?

[bunny]

Thanks for sharing