cell death during transfection - (Jul/10/2006 )
So I have been transfecting primary fibroblasts...and my plasmid only and lipofectamine only wells have little to no death....I only see death where there is DNA and Lipofectamine....so .....do you think that perhaps there is something specific about the cell becoming transfected that is causing the death?? Have any of you had this problem??
Ames
Ames
I have seen a quite a few dead cells after transfection with lipofectamine. but it depends on the amount of lipofectamine being used. If I use less of lipofecatamine then the amount of dead cells is also less.
I've seen this happen, particularly transfecting primary fibroblasts with BAC DNAs or low copy plasmids which required a high culture volume for a small amount of DNA.
Make sure your DNA is very clean and check it on a gel to make sure it's undegraded. If you have endotoxin (which you wouldn't see on a gel) or bacterial chromosomal DNA, these could be toxic when included with lipofectamine to enter the cells. Try reprepping the DNA with a commercial kit, GeneCleaning (or other such clean-up kit) the plasmid you have, or phenol:chloroform:IAA extract it and EtOH precipitate it. Follow the protocol for your lipofectamine reagent carefully, especially with respect to DNA and lipofectamine masses and volumes.
The gene product form your plasmid could also be killing the cells, but primary fibroblasts (human, anyway) transfect poorly so this would only explain 10 - 20% of your cells dying.
Try a control plasmid (EGFP driven by the CMV promoter) made by a reliable source + your lipofectamine to test your cell death and transfection efficiency.
Unfortunately, cell toxicity comes with lipid- or polymer- based transfection reagents. It is well known that lipid/DNA complexes are more toxic than individual components. Some transfection reagents are more toxic than others. The one that you are using is a potent one in terms of transfection efficiency as well as toxicity. You just have to expect some level of toxicity. In some cases, those cells received most complexes get killed. Up to 50% maybe killed at the end. I agreed with previous two members, the dosage of lipid/DNA and the quality of DNA seem to be the key factors. Primary cells will be more sensitive to LPS contaminant.