Problems with transfection in human cells (View forum version)



AbelMartin

Posted 26 February 2011 - 04:02 PM

hi everyone,
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.
Thanks

protolder

Posted 28 February 2011 - 10:39 PM

Hola, my experience with human cells is very limited, but there is an special kit of plasmid purification from Quiagen to achieve endotoxin free plasmid to transfect sensible cells, that seems that is your problem. I hope this could help you. Buena suerte

AbelMartin

Posted 01 March 2011 - 11:22 AM

Hola, my experience with human cells is very limited, but there is an special kit of plasmid purification from Quiagen to achieve endotoxin free plasmid to transfect sensible cells, that seems that is your problem. I hope this could help you. Buena suerte

Hola, I already use this kit endotoxin free from Qiagen. I was thinking that this could be the problem but I also use GFP from Amaxa which is endotoxin free and I have the same problem.
Thank you for your suggestion.

apeters

Posted 02 March 2011 - 02:17 AM

HI AbelMartin,

Nanotherics (google search them) have a protocol for difficult to transfect cells using nanoparticles. Might be worth a try?

APeters