Hi,
I am going to use ClearView 96-well plates from EssenBioscience to do migration assay using IncuCyte, similar as transwell boyden chamber. My question is whether I can do reverse transfection on the chamber? In another word, add basic media and siRNAs plus transfection reagents like RNAiMAX from lifetechnology, onto the membrane, then add cells to the mixture media containing siRNAs. Then culture for 2 days to let siRNA take into effect. Then on 3rd day, replace the top part with serum-free media and bottom part with complete media, to actually run transwell migration assay.
The reason for putting in this way, is that, I am screening on 40 siRNAs, to see their effect in migration... If I knockdown the gene in 6-well plates, I have to calculate the number of cells after two-days of knockdown before plating them onto the chamber... calculating cells will be too much work and is very low throughput...
I have been using this reverse transfection in measuring proliferation, which is very fast... so I am wondering whether someone has done reverse transfection with boyden chamber?
Many thanks!
If you're only testing 40 siRNAs, I would recommend using live microscopy to assess the effect of knockdown on cell migration. In my opinion, live microscopy provides a lot more information on cell migration (speed, directionality, persistence, cell morphology) than a transwell assay.
I guess you can't do the transfection directly, because you will always keep the effect on proliferation and thus different cell numbers....
best of luck!
Hi!
If I was in your case, I would just give it at try. Do you plan to use siRNAs that you have already tested for their effect on viability and they don't change much? This is an important prerequisite to be able to generate reliable results.
If yes, I would do a first test using only your control siRNAs where you also make sure that there is no basal migration before your medium change. This will - according to my experience - depend on the cell line you will use.
Good luck,
Cindy
Try Chemotaxis assay from Essen Bioscience. Currently this is the best option on the Chemotaxis assays market: http://www.essenbioscience.com/applications/cell-migration-chemotaxis/
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