We are trying to transfect human fibroblast that are typically slow growing and can take a little while to get up to 70-80% confluency. We do try to plate them at a specific density, but sometimes that is not possible. so we have to wait for them to reach the optimal confluency, which sometimes can take a week. Is it okay to transfect cells a week or more after seeding them, as long as they aren't too confluent?
Hello Amanda Marie
It is not okay to transfect cells a week or more after seeding them. The transfection efficiency may decrease. The maximum time frame you can wait to transfect cells after seeding cells is 24 hours.
Cells should be plated 18–24 hours before transfection to ensure that the cells are actively dividing and reach the appropriate cell density (generally 70–80% confluence) at the time of transfection. For higher transfection efficiency, the cells should be > 85% viable at the time of transfection and they should be in early passage.
You should perform a pilot experiment with different seeding densities with the aim of obtaining 70-80% confluency in 24 hours. Then proceed with the transfection experiment using the optimal seeding density.
Best.
Have you looked up at topics around transfection optimization? I have dealt with this with my PhD thesis. There are many parameters (confluence, ratios, DNA input etc). Most importantly, I would check for mycoplasma contamination. In fact, don't even bother doing any of this if you don't know the mycoplasma status of your cell stocks. It is a subtle contamination that doesn't behave like your typical microbial contamination - no pH/phenol red changes, no smells, no turbidity etc. Mycoplasma can have severe inhibitory effect on transfection, I would know because we've seen it first hand. Everybody thinks they don't have mycoplasma contamination - until they test :).
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