I would like to perform a migration assay and then isolate only the migrating cells (the ones that reach the other side of the membrane!), in order to plate them again and do other tests on them. Has anyone ever tried that? What is the most effective way of isolating those cells and keeping them alive for more analyses? Any suggestions?
Hi,
I never did cell isolation however I have dealt several times with Boyden Migration assays and went through literature. I would suggest to remove the non migrated cells using media wet cotton bud and gently dipping the insert in 1X PBS and placing them in further diluted 0.25% trypsin or Versene-EDTA for few minutes( time might needs optimization depending upon the cell types). In brief follow the normal protocol of trypisination or splitting of the cells. The limiting factor should be number of cells you get at the end of isolation. For which instead of 24 well, 6 well or 12 well format should give better results with respect to number of cells. The cells can be expanded and used further for assays. I hope this might be of some help.
Best wishes,
Dinesh
Thank you very much, I was thinking about something similar!! I will give it a try!!
I have done this successfully by doing an inverted Matrigel assay. I first set the Matrigel in the transwells and flip the 24 well plate over so the transwells stand upside down on the lid. Then I seed the cells on the bottom side of the membrane and allow them to attach. I flip the assay over again and add chemoattractant at the top side and low serum and no attractant to the bottom compartment. After three days I wipe all the non-migrated cells off the bottom using a tissue with ethanol. I then cool the plate in the fridge for 30 min which will dissolve the Matrigel and then you can collect the cells that have grown in the Matrigel easily for further purposes. I do keep them generally in 1:10 diluted Matrigel to have a 3D environment. This design is limited to a 24 well plate, but I generally get quite a few migrated cells out of this that I analyse with FACS, western blot etc.
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