Recently, I am working with huamn vascular smooth cells and human dermal neonatal fibroblast. I have to transfect my cells with differents plasmid. In order to improve the technique I was use GFP control (differents vectors just only carry GFP). I use Mirus or Amaxa as transfection method. When I transfect my cells during the first 24 hours or overnight (in serum) they look fine. Due that I work with TGFb, I need to preincubate my cells in serum free for 24 hours (after the initial overnight in serum) and then treat in serum free for another 24 hours (so in total 72 hours, 24h in serum and the last 48h in serum free). Exactly after the treatment with TGFb I see that after the last 24 hours I have a cell death just only with plasmid (not with reagent alone), and more important my cells do not respond to TGfb when they had been transfected with plasmid (also in not GFP plasmid). However cells just only treat with the reagent or transfected with amaxa are ok. Moreover, if I use siRNAa with Amaxa they respond perfectly. My guess is that the plasmid is doing something. Has anybody had similar experience?, any suggestion?.
I really need to know if there is an alternative or is common since the luciferase experiment that I am planning to do is critical for my project and I do not wanna have wrong results due to lack of responsiveness of TGFb in my cells.
Edited by AbelMartin, 26 February 2011 - 04:06 PM.