Abstract
Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, usually a tissue-culture-treated microporous membrane, which is positioned between two compartments that mimic two different sets of microenvironments for cell survival/growth. Cells on one side of the membrane, when sensing chemoattractants placed on the other side of the compartment that diffuses through the membrane, can migrate through the pores in the membrane towards the source of the chemoattractants. Cells that migrate across the membrane can be quantified by fixing and counting. Human breast epithelial adenocarcinoma MD-231 cells grow relatively fast and are metastatic. The MB-231 cell line is used here to describe the procedures of an in vitro cell migration assay using the transwell apparatus.
Materials and Reagents
Equipment
Procedure
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
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
There is nearly no cell in your picture. Maybe lengthen the time of culture or put more cells may help.
This experimental setup allows gradient of chemoattractant be established, which is the "driving force" for migration. We don't know how long it takes to "equilibrate" if that would happen eventually, time is also a determining factor here, I think.
Collagen is added as a chemoattractant; you may use other chemoattractants of your choice.
Thank you!I have another question. Is the 2% ethanol in crystal violet solution necessary? If I use the solution that crystal violet dissovled in ddH2O, can it work?
I guess that should work too as long as you can get it dissolved well.
1. Please make sure that the bottom of the insert, when immersed in medium, has no small bubbles trapped underneath (especially the middle area). The microporous membrane should be soaked evenly in medium.2. Please add the volumes of media/cell according to manufacturer's manual, which keeps the inside/outside media at certain levels.3. Avoid swirling medium when adding cells to the insert.4. The transwell plate should be kept steady when incubated, reduce moving it around or shaking it.5. For your experiments, you may also way to try different cell seeding densities, and/or a shorter migration time.Hope these can help.
In the author's hands, EGF has never been required for successful migration assay using MB231 cells. Some researchers even carry out this assay using medium without either FBS or EGF. Please see reference: PLoS One. 2010 Dec 30;5(12):e15940.