Materials and Reagents

Human MDA-MB-231 cell (ATCC, catalog number: HTB-26 ™)
Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
Trypsin-EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056 )
Trypsin inhibitor (soybean) (Life Technologies, Invitrogen™, catalog number: 17075-029 )
Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
Collagen I (Sigma-Aldrich, catalog number: C7661 ) or Fibronectin (BD Biosciences, catalog number: 354008 )
Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
Ethanol (Sigma-Aldrich, catalog number: 459836 )
Crystal violet (Sigma-Aldrich, catalog number: C3886 )
TCC-formulated Leibovitz's L-15 Medium (ATCC, catalog number: 30-2008 ™)

Equipment

Corning^® Transwell^® polycarbonate membrane inserts (Sigma-Aldrich, catalog number: CLS3421 ) or Millicell Cell Culture Inserts (EMD Millipore, catalog number: PI8P01250 )
Cotton swabs
Cell culture incubator: 37 °C and 5% CO₂

Procedure

Carry MB-231 cells in DMEM with 10% FBS (use L-15 medium if needed).
Wash cells twice with 1x PBS and trypsinize.
Add 0.5 mg/mlTrypsin inhibitor in PBS to inactivate an equal volume of Trypsin. Aspirate cells by pipetting up and down gently (Note: It is important to break down into individual cells as much as possible).
Gently spin down the cells. Wash cells two times with DMEM containing 0.5% FBS to remove trace amounts of trypsin and inhitibor. Resuspend the cells in DMEM with 0.5% FBS and count.
Prepare the transwell compartments, 24-well format, with 8 μm pore size insert:
1. To the lower compartment, add 2.6 ml of DMEM with 0.5% FBS containing 40 μg ml^-1 Collagen I.
2. Add the transwell insert to the well by merging the bottom of the insert into the medium in the lower compartment.
Note: Ensure that no air bubbles are trapped between the insert membrane and the medium.
To the upper compartment, gently add 1 x 10⁵ cells from step 4.
Incubate the cells in the transwell plate at 37 °C and 5% CO₂ for 2.5 h. This allows cells to migrate toward the underside of the insert filter.
After 2.5 h, carefully take the insert out. Cells that do not migrate through the pores and therefore remain on the upper side of the filter membrane need to be gently removed with a cotton swab. Gently wipe the upper side of the filter membrane with a cotton swab to remove the cell debris. We recommend that use each clean cotton swab for one wipe only, in one direction and do not swipe in back-and-forth movement. The cotton swab can be slightly moisturized with ddH₂O as needed but be sure to remove any excess water. Several wipes may be needed to completely remove any cell debris on the membrane.
Fix the cells on the lower side of the insert filter quickly with 5% glutaraldehyde for 10 min.
Next, stain cells on the lower side of the insert filter with 1% crystal violet in 2% ethanol for 20 min.
Remove excess crystal violet by quickly merging the insert in ddH₂O for 3 to 4 sec. Drain excess water from the side of the insert using a cotton swab. Dry the insert membrane.
Count the number of cells on the lower side of the filter under a microscope. Randomly choose different views and take average counting.
The same experimental procedure should be performed for control groups without chemoattractants. Each migration condition should be tested with replicates.

Acknowledgments

This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Katoh et al. (2006) and Nakamizo et al. (2005). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee [see Chen et al. (2009)].

References

Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
Katoh, H., Hiramoto, K. and Negishi, M. (2006). Activation of Rac1 by RhoG regulates cell migration. J Cell Sci 119(Pt 1): 56-65.
Nakamizo, A., Marini, F., Amano, T., Khan, A., Studeny, M., Gumin, J., Chen, J., Hentschel, S., Vecil, G., Dembinski, J., Andreeff, M. and Lang, F. F. (2005). Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 65(8): 3307-3318.