Hello everyone,
I am currently having problems with my transfections and request help. I use lipofectamine 3000 to transfect Hek 293 cells in 6 wells plates. For the last transfection, I transfected at 60% confluency. Also, I used 2500 ng of DNA per well, 5µl of P3000 per DNA, 5µl of Lipofectamine 3000, and DMEM with P/s + L-glu instead of OMEM. However, after 48 hours of transfections majority of the cells were lifted off the plate. However, even though the cells detached from the plate and little pellets remained after they were centrifuged, the lysate showed protein expression but not the medium (shown from western western analysis). But the problem is that I need protein to also express in the medium. Even when I incubated the transfected cells for 72 hours and used OMEM, there still isn't protein expression shown in the medium.
Any advice or recommendations will be greatly appreciated.
We sometimes transfect the cells at multiple passages, and it seems to give better results. For Hek293, try this kit, which is optimized specifically for Hek293 cells (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/).
Hi Vanessa,
You should increase the concentration of DNA per well.From my experience I increased it to 5000ug per well which increased my Transfection efficiency.Rest of the components can remain the same.Also , sometimes while pipetting DNA/lipofectamine complexes in the wells, the attached cells get disturbed which leads to their detachment from the surface.
Hi Nitika,
Thank you for help! I'll increase the DNA concentration for the next transfection and be very careful during pipetting.
why dont you use media without P/S ? i heard that P/S like antibiotics may disrupt transfection efficiency. so when i transfect to cell, i usually use Opti-MEM or 2% FBS in media without P/S. i assume that it may help your experiment
and also have you ever concentrated you supernatants? there are little proteins in large volume of medium, so you should concentrate with kit or other method to your supernatants..
Hi Vanessa
I transfected Hek293 with more than 90% efficiency. my suggestions are:
firstly do not use P/S at all while your are transfecting cells
secondly, it's very important that you transfect the cells which are at second or third passage so they are stable. don't transfect lower or higher passages of the cell
you also can increase the time of incubation of the lipo/P3000 combination before transferring it to the wells .
Hi Rak-hyun, When i end the transfection, I concentrate the medium using 10,000 MWCO tube for 10 minutes (1800 rcf in 4C) but there was no protein expression in the medium. However, for the next transfection, I won't use p/s in the medium. ill just use DMEM without FBS, P/S and L-glu when diluting the DNA and LF3000 but use DMEM with 2% FBS during the transfection. Thank you for the advice!
Hi Shabnam, I followed the thermofisher protocol which recommends 5µl for P3000 but I'll decrease the amount to 4ul and use the same amount of LF3000 (5µl), but is it wise to use more LF3000 than P3000? Also, thank you for your help!
Hi Maryam, I won't use P/S during the transfection. Ill only use DMEM with P/S+L-glu when seeding the wells. Also, Ill transfect the cells at 80% confluency. Also, I usually incubate the DNA-lipid complex for 15 minutes and then add it to the cells, but I'll wait 20 minutes. Thank you for your suggestions!
I know for a fact that using P/S during a lioidectamine transfection increases toxicity massively. 2500ng DNA 5ul of P3000/Lipo and using optimem to form the lipid complexes will solve your issue. These details usually result in an almost 100% transfextion efficiency with minimal toxicity. Good luck
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