Hello all,
I am using HEK293T and HEK293-Peak Rapid cells, 0.01% PLL, Tryple Express, Optimem with 4% FBS, Lipofectamine 2000, and plasmid on 96-well plates for a transfection protocol leading into a calcium mobilization fluorescence assay and repeatedly run into the issue where after 4-6 hours of transfection the cells lose their healthy morphology/filopodia and detach from the wells. Some cells remain but not enough to yield good, consistent results.
I'm coating my plates with the PLL for 1 hour, sometimes longer. I'm then washing those plates with sterile water. I'm plating 5,000-25,000 cells per well with Optimem + 4% FBS, then transfecting 20-24 hours later. I replace the Optimem + plasmid medium after 4-6 hours with fresh Optimem with 4% FBS until the assay the following day. Between the end of the transfection and the following day when I am ready to begin my calcium mobilization assay, the cells change their morphology, become round, and detach. For the few cells that do remain, I get a fluorescent response to ATP and some ligands to transfected receptors. The only issue is that not enough cells remain to reproduce consistent and reliable results. Has anyone else experienced this? What can be done to fix this issue? Any help will be greatly appreciated. Thank you.
A couple of possibilities:
1. There are too many cells in the well and they are overgrowing and coming off. Maybe plate on the low end (5000 cells) rather than on the high end.
2. Do you normally grow the cells in 4% FBS or you use 4% for just for this assay? They may not like the reduced FBS condition if they are used to growing in a higher amount.
3. Perhaps 4-6 hours in the transfection media is too long. I never go more than 4 hours because the cells start to look sickly. How do the cells look after removing the transfection media?
Tom Masi Thank you for your response. I will try to see if using fewer cells produces better results. The cells are typically grown in DMEM with 10% FBS, so that is a possibility as well. As for the transfection duration, cell morphology begins to change after transfection and the process of removing and replacing medium is when detachment begins.
You can adapt the cells to grow in 4% FBS. They will grow slower but still function normally. I have adapted mine to grow in 5% so I don't have to deal with them as frequently. My guess is the time of transfection is where your problem is especially with Lipofectamine. I use PEI for transfection and it doesn't seem to be as toxic to HEKs but I still stick to the ~4 hour mark for time.
Tom Masi Awesome. I'll give this a shot. Thank you very much Tom, you were extremely helpful.
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