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70% of scientists have extremely high expectations regarding the accuracy of their DNA oligo shipments
--Oligonucleotides: Uses & Applications | | | | | Protocols
Welcome to The Science Advisory Board's extensive database of research protocols for the biomedical sciences. Protocols are organized by techniques and are fully searchable by key word(s). Protocols are divided into a brief description of the methods, a step-by-step overview of the procedure, a collection of recipes, the supplies needed, and any helpful tips. Members submitting protocols will be rewarded through the ViewPoints system. ... back to articles list Matrigel invasion assays Posted on Saturday, October 18, 2003
Description
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.
Procedure
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.
Recipes
- Matrigel (Becton-dickinson)
- 24-transwell (Coster)
- Fibronectin(Sigma)
- Diff-Quick staining solution (Fischer Scientific)
Supplies
Tips
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