Hi All,
I've been reading this forum for ages and have got some really useful information. I've joined up to ask for some advice about transwell migration assays...
Six months ago we were routinely performing migration assays and getting excellent results. However, we have recently restarted using this assay and are having major problems. It seems that we are getting very little migration. We were previously using Corning 24-well transwell inserts, however we changed to BD Bioscience chambers instead as they are a lot cheaper.
We serum starve the cells (cancer cell lines), trypsinise, count and add 100,000 cells (in serum-free DMEM +0.1% BSA) to the top of the chamber (8um pore).
We use DMEM+ 20% FBS in the well to attract the cells.
We incubate for 4 hours, then fix in 100% methanol, stain with 0.1% crystal violet and count cells in four fields of view.
There seem to be a lot of cells around the edge of the membrane (it looks like a halo), but there are no cells migrating through the main surface of the membrane.
At first I thought it was my cells, so I have used 3 different lots from the liquid nitrogen and also a different cell line. None will migrate through the membrane.
Any advice would be appreciated as I'm starting to pull my hair out!
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