Dear collegues, does somebody use PEI to transfect cell lines? We are having a very low efficiency (less than 50%) and we need a higher transfection rate for the experiments. Do you know how to improve this technique? Thank you very much.
Coat the plate with poly-L-lysine for 20 min. Then proceed for transfection.
Hi Favio,
There is a technique I used to improve almost every transfection reagents' efficiency.
After trypsinization, pellet your cells (1000rpm), discard the medium and resuspend them in your mix (PEI+DNA). Put them in your water bath so they rapidly reach 37°, for 10min. Then you can plate them in your dish.
Hope it will work better.
We use jetPEI in our lab. It's a proprietary PEI solution from Polyplus that uses a very specific molecular weight PEI, as opposed to the heterogeneous solutions that are normally supplied as "PEI". It's a little more expensive than PEI, but definitely cheaper than reagents like Lipofectamine and we have had good yields with little optimisation.
To put a number on "a little more expensive": 4 ml of (maybe 1 mg/ml solution) JetPEI are about 800€ (here in Finland). 100 ml of PEI (Sigma 408727), which has been used for transfection e.g. in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630511/pdf/nihms-456892.pdf are about 80€. Hence, jetPEI is more than 10000 times more expensive than the Sigma stuff. We have tried both and JetPEI works more robustly and reproducibly. However, we have gotten quite good results with the cheap stuff as well, but we are not sure why the transfection is much less reproducible...
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630511/pdf/nihms-456892.pdf
We are not able to spend that money for a reagent. Hence, we will try the technique of David. Anyway, thank you very much for all your answers.
Hi Favio,
we have also increased transfection efficiency (using the cheap PEI) with the trick mentioned in "LabTimes". Seems that everything helps which increases the number of collisions between cells and DNA complexes. A high concentration is very important (David's method) and agitation as well. However, if you overdo it, you may kill the cells both by too high concentration and too much agitation. But that is of course very much dependent on what cell line you use...
http://www.labtimes.org/labtimes/trick/tricks/2010_04.lasso
David Müller I would suspect that might aggravate some cell lines. Do you remove the PEI + DNA mix after they adhere? Also what is the ratio of PEI + DNA to cells that you use? I'm going to try this technique!
The DNA/PEI ratio is confusing in the literature since everybody defines it differently. We have used the definition from Zhang et al. 2003 (Article Polyethylenimine strategies for plasmid delivery to brain-de...
) and we had best results with high amounts of DNA ( >2µg DNA per one well from a 6-well-plate) and N/P (PEI/DNA) ratios of >1. The take home message is to use as high N/P ratio (as much PEI as possible) and as much DNA as possible without killing your cells. I guess you should do an optimization first (trying out ratios between 0.2 to 10 and DNA amounts from 0.5 to 5 µg for a single well on a 6-well-plate). However, we have problems with the reproducibility (once the transfection works well, next time not so much) and that problem seems to have been solved by the Polyplus company (we keep using JetPEI). However, we used the neutralized PEI (pH 7) and some groups report that the acidic PEI works better and more reliably...
We use approximately 3 μg of DNA and 3 μl of PEI in a 35 mm dish. My consultant tested different concentrations of DNA and PEI, and this was the optimal one to transfect HeLa cells. However, the reproducibility is not so good. We use PEI to transfect all cell lines, except the neurons with which we use Lipofectamine. I have not yet been able to test higher concentration of PEI, like the method proposed by David Müller
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