stage at which transfection should be done, when cells are round or flat adhered - (Oct/31/2007 )
I want to do stable transfection of H295R cells with lipofectamine 2000. But I have a concern that generally people say that the day after you seed cells do transfection. But my cell line is very slow and cells remain round even after one day. So what should I do wait for cells to become flat and adhere to the bottom of the culture plate or I can do transfection next day. When should one do transfection on the round cell or flat cell.?Please guide me.
Thanks in advance
Usually people wait for cells to be 60-70% confluent before transfection. I donot know about them being flat or rounded. Transfect some wells and check it.
Cell number can be critical for the amount of toxicity. More toxicity is expected if you dont have enough cells to take all the reagents.
I used scolix's protocol doing transfection while 293 cells are in suspension. It worked very well.
You dont pre-seed the cells. Just mix the transfection complexes with the cells in suspension then plate them.
If this protocol hold true to your cell type, then the timing after plating would be less important. Why dont you go ahead and try it and let us know what happens.
I am also interested in this kind of protocol? genehunter or scolix is it ok to write it down?