Essentially, we have various HCC cell lines that we plan to do a migration assay using corning transwell 8.0 um pores w/ matrigel. Utilized a chemoattractant of 5% FBS/DMEM serum as the chemoattractant. Utilized some growth factor + inhibitor that was present in other wells for variation. We seeded 50,000 cells and they were visible in the insert following seeding. However, when I washed/scrubbed the insert following fixation (4% PFA, then 1% crystal violet soln). The issue comes when I observed the cell/inserts under the microscope for imaging. I noticed that there were only cells present around the edges of the insert..My assumption is during the scrubbing with the cotton swab, the cells were removed. In this assumption, I would then assume that the cells never migrated down through the pores. For the chemoattractant with the growth factor, we used a concentration of 50 ug/mL and on the last variable, we used an inhibitor following that pathway with a concentration of 1.0 uM. The cells were allowed to migrate for ~24 hrs. I suppose, we could increase invasion time to 36 hrs to see if that works, but not sure what else to troubleshoot, as scrubbing is necessary to remove non-invaded cells..
Thanks in advance for the assistance.
Hi Danisha,
what also puzzles me is that you obviously see migrated cells, however those are only located at the periphery. To me that really sounds like you have a problem during the removal of the cells on te top side of your membrane?
Maybe it is just that you simply apply too much pressure with the swab during the cell removal step and that also affects the cells that have actually migrated through the membrane?
This is invasion assay actually. Migration assay would be free of matrigel.
Except for what have mentioned by Abderrahman, which I strongly agree with, here are something you may want to try.
1. Coat the transwell with collagen.
2. Increase your cell load.
3. Try scratch assay in parallel.
Hope this can help.
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