HEK 293T Transfection problem
Posted 20 August 2012 - 07:14 AM
I'm new to doing transfections. I have a special cells line lenti-x 293T that is an HEK 293T derivative supposedly optimized for transient transfections. I followed the protocol for GenJet online which suggests transfecting at 60-70% confluency and harvest 3 days post transfection. However, after a single day, I've noticed that there's a lot of dead cells and debris in the media, and that the cells already look overgrown and somewhat apoptotic. Should I be worried about proteases from dead cells? Is this a usual phenomenon to see dead cells?
Posted 20 August 2012 - 02:06 PM
If you are using PEI, change medium after 6h - should help.
I also use more confluent cells: 80% - 90%.
Posted 20 August 2012 - 02:38 PM
I wouldn't be worried about the proteases in the medium, unless you are looking for a secreted protein that you hope to harvest from the medium.
293-T are extremely easy to transfect, they are one of the more commonly used cell lines for this purpose. They do have the drawback that they are only weakly attached and can, and often do, float off with knocking of the flask, pipetting directly onto the cells, and treatments with chemicals (including TRs).
Your lenti-x cells are probably best for production of lentiviral particles, rather than just transient transfections.
Many other cell lines are very good for transfection, depending on what you are doing, another cell line might be a better choice for transient expression.