Jester4023

I've been trying to get wound scratch assays working in cells transfected with siRNA but am having problems getting the cells confluent enough.  I'm doing reverse transfections using RNAiMAX as my cells are difficult to transfect and normal forward transfection doesn't seem to cut it.

I've done plating efficiency to figure out how many cells I need to plate in order to get a confluent monolayer to scratch, but the cells don't seem to spread out after transfection.  They'll adhere, but remain quite rounded.  I've tried different cell concentrations, but still the same problem.  There are plenty of cells there, but they're all rounded up and not filling in the gaps on the plate.

I've tried setting up the transfections in larger plates and trypsinizing and replating them into the scratch plates when you would normally put post-transfection media on.  I've tried putting the larger plates into fewer, smaller wells for the scratch but that's not working either.  I finally got what looked like enough cells and a nice monolayer today, scratched the plates and went to image them and all the cells had washed off the plates.

I can't really leave the plates much longer before I scratch because I run the risk of missing the knockdown window from the siRNA.

Any suggestion at all would be greatly appreciated!
Thanks
Jess