Not sure if anyone has had this problem. We work with a primary cell line that we've been trying to transfect with pcDNA3.1. I've been using Lipofectamine 2000 to do the transfections and leave it on for 4 hours, then wash with PBS and add free media containing serum. The next day after transfection, the cells that were transfected with vector alone or vector containing my transgene are looking retracted compared to the Lipo only controls. Then when I use the cells in a spreading assay, the Lipo cells spread just fine, but the cells transfection with the vector or transgene only stick and stay round. So far we've tried using different ratios of DNA to Lipo and they seem to look better at a 1ug DNA to 1.5ul Lipo ratio, but still do not spread. Are there any other ideas for troubleshooting? My boss wants us to check the endotoxin levels of the plasmid DNA, but I'm not sure that's the problem. Help?