Hi Srijan

I think the PEI:DNA ratio you used is too high, I suggest you to try 2-2.5 ratio. Besides, the cell state and confluence are very important for a high efficiency transfection, you should replace medium before transfection and ensure the cell confluence between 70-80%.

Genemedi has launched a transfection reagent of Lipogene, which is comparable to lipofectamine 3000, much more effictive than PEI. You could find more information on this website: www.genemedi.net/i/lipogene-transfection-reagent

Hope this would help you.

In my experience, HEK293 is very easy to do transfection with high efficiency. Why dont you do CaCl2, lipofectamin 3000, or electroporation? Using CaCl2 is very good and cheap way to do transfection. 

If you use PEI, it is also cheap and high efficiency of transfection. In your case, I recommend you to check again your DNA. Is it really purity. DNA concentration could affect the transfection efficiency. Did you use mi-di prep to prepare your DNA?

I would also recommend checking your DNA. You could also try to increase the DNA amount or to increase your 4h incubation time.

Dear Srijan,

as mentionned by Trang, the conditions seem ok but Sophie and Daniel are also right. Actually there is no rule at all for transfection efficiency: depending on the cell type (clone), plasmid design, plasmid purification (presence of salts),  level of coil, strength of the promoter, medium, kinetics of experiments, read out, (...) efficiency can vary from one lab to another.

As control, I would recommend you trying an alternative: our DreamFect transfection reagents. They have been published by many authors to be real efficient on many cells (of whom HEK-293).

As an example, you can refer to our publication database following the link below.

Would you need a sample, or would you have any question please do not hesitate to contact me directly at: [email protected]

best regards,

Cedric

http://www.ozbiosciences.com/module/citationfinder/default

Dear Srijan,

I usually transfect HEK293 cells and the incubation time that usually works for me after transfection is 48 hours. And also, before transfection don`t leave your cell grow too much, usually a 65% confluency is the best when transfecting HEK293 cels.

Regards

Darlène

In my recommendation you should use lepofectamin and 24 well culture plate for s. B.Coz efficiency will be more if you transfect in 24 well plate.

Are your cells getting a bit old (high passage number)? I don't have any solid data to back it up but I have observed that 293Ts don't transfect as well when they are high-passage, especially if they're 'clumpy' as well

You should check the purity and concentration of the DNA. I always use lipofectamine 2000 for transfection of HEK293T cells. The cells should not be old, preferably 1st to 4th passage is good for high transfection efficiency. Also you should increase the DNA amount. I use 10 ug DNA for 100 mm culture plates and 3 ug for 6-well plates. The cells should not more than 60-65% confluent during transfection.

I split 80% confluent HEK293T 100mm plate into 4 100mm plate 16 hours before doing transfection. For me, FuGENE 6 from promega is giving 90% transfection efficiency. But again it depends on your plasmid. I use 8 ug of plasmid DNA with 4 ug of packaging vector with opti-mem and FuGENE 6 to produce retro virus. 48 to 72 hours after transfection gives the best result. Hope this helps.

I used to do HEK293T transfection with 25 uM of Chloroquine with CaCl2 protocol. We added the chloroquine 5 minutes before transfection. The other protocols explained here are good for HEK293T transfection.

Meybe, you can precipitate with isopropanol and AcNa 3N  your plasmid to clean it. That is important for a good transfection.

good luck

Hi Srijan,

I may have a good solution for you. A chemist (Dr Pavel Levkin) and myself (Biologist) are both group leaders at KIT and started an interdisciplinary project almost 3 years ago that lead to the discovery of novel lipid formulations best suited for HEK cell transfection. I use HEK cells routinely for large scale screenings so I had an interest in such development as lipofectamine was too expensive for our lab!. We now commercialize ScreenFect reagents through the KIT spin-off company Incella.

We can supply our reagents for much less than Lipofectamine (our list price for 1 mL is € 175 but we offer 50% discount to all academic research users willing to share their transfection data with us). That's only € 87 for 1 mL (compared to over € 500 for same amount of Lipofactamine).

We have optimized HEK and HeLa cell transfection using the so-called 1-step protocol that saves one day because it does not require pre-plating of cells the day before transfection. Simply perform the transfection on freshly detached and re-suspended cells. This allows you to perform a transfection when splitting your cells. It works far better then Lipofectamine and is less toxic!

We can send you some complimentary test samples of our 2 transfection reagents for HEK cells, which are ScreenFect A and ScreenFect A-plus. Just let me know.

best,

Gary

Dear Srijan,

the optimum for PEI, DNA and cell concentration is different for each cell line and medium. You can optimize your protocol by trying different DNA, PEI and cell concentrations for the transfection. In many cases a DNA:PEI ratio of 1:2 or 1:3 works well. DNA concentrations frequently are between 1-2.5 mg/l. You can start with 1 mg DNA per 10^6 cells.

We developed a suspension growing HEK293 cell line (MEXi, http://www.iba-lifesciences.com/newsdetails/items/mexi-mammalian-expression-system-from-iba.html) with affordable transfection and cultivation media and optimized the protocol for PEI transfections. We found that a ratio of 3:1 (PEI:DNA) was the optimum for our system using a DNA concentration of 1.5 mg/L and a cell concentration of 1.5 x 10^6 cells/ml. The toxicity was very low at a final PEI concentration of 4.5 mg/L. The viability of the cells is > 90 % 24 hours after transfection and cells recover without any medium exchange after the transfection. The cells can grow from 7.5 x 10^5 cells/ml to over 10 x 10^6 cells/ml.

Best regards

Dennis

 Dear Dennis,

I would like to ask about the procedure to convert adherent HEK293 to suspension cell line.

I have adherent HEK293 cells but i read about its transfection and come to know that suspension cell of HEK293 are more efficient for transfection.

Thanks

Jyoti

Dear Jyoti,

there are two strategies to adapt cells to suspension. The quick one is to detach the cells (e.g. with Trypsin) centrifuge them and re-suspend them in a medium which allows efficient suspension growth or thaw the cells in the medium directly. Usually this media contain no or a reduced amount of serum. After re-suspension you can culture the cells on a shaker.

The longer procedure is necessary if the first one does not work. For some cells and media it is not possible to remove the serum completely in one step. In this case you can add serum to the serum free suspension medium. For every adaptation step you would reduce the serum concentration by 50 % and culture the cells until they show the same viability and doubling time as before. Then you reduce the serum concentration again by 50 % and so on. You can also try to mix your serum containing medium with the serum free medium and reduce the portion of the serum containing medium successively.

However, it can take time and effort to find a media which allows good suspension growth of your cells and to adapt the cells.

We offer a HEK293-System consisting of suspension growing HEK-293 cells and serum free media. You can use the system without the need to adapt the cells. There are no license fees to pay for the use of the system, doesn’t matter if you use them for R&D or for commercial use.

Furthermore, we offer several Vectors which are matched to the system and our tag based purification technology. We provide everything you need to come from your gene of interest to purified protein, so you do not need to develop a production system by yourself.

You can find more information on http://www.iba-lifesciences.com/mammalian-expression-technology.html

I hope I was able to help.

Kind regards.

Dennis

Dear Dennis

Thank yo so much for this valuable information.

With regards

Jyoti

Dear all,

if you are looking for media and protocols for efficient transient expression of proteins in HEK293 or other human cell lines, I highly recommend Xell's HEK TF media for use with polycationic transfection reagents. You can use the same media for your seed, for expansion, transfection and production. If you want to boost your protein titers further, you can easily run a fed-batch by adding our matching HEK FS feed supplement as required.

http://www.xell.ag/products/#culture-media-and-feeds

Also check out our poster on this topic:

http://www.xell.ag/doc/ESACT_2013_HEK_Poster.pdf

Do not hesitate to contact me or any other member of our dedicated team directly, in case you have any question regarding our media or protocols.

Kind regards,

Frederik

Hi, I suggest Nanocin reagent for this, which works great with HEK293 cells, L

Although HEK293 should be pretty easy to transfect, but if you're having issues try using a HEK293-specific reagent (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/). That should ensure that you get better results in vitro.

Dennis has great recommendations for getting the cells into suspension. I have had great success going into ExCELL 293 medium which contains hydrolysates directly from serum containing adherent cultures. One key that I've seen is that when you first go to suspension, agititate them super slow for the first couple of days - 60 rpm on a 19 mm throw platform Once they start growing, increase the rpm to 120.

Once they are growing well, I have also found that you can then switch to a chemically defined medium.

Sandy

In addition to optimizing DNA preparation and changing PEI to other transfection reagents, probably you need to think whether and why the expression level of this protein should be high.

Don't change the media after 4h, let your cells incubate with the transfection mix overnight.

Good luck!

Hi Srijan

I think the PEI:DNA ratio you used is too high, I suggest you to try 2-2.5 ratio. Besides, the cell state and confluence are very important for a high efficiency transfection, you should replace medium before transfection and ensure the cell confluence between 70-80%.

Genemedi has launched a transfection reagent of Lipogene, which is comparable to lipofectamine 3000, much more effictive than PEI. You could find more information on this website: www.genemedi.net/i/lipogene-transfection-reagent

Hope this would help you.

I also have question involved with transfection using PEI. In my experiment, I try to transfect my DNA to SY5Y and 293T cells using PEI. In 12-well plate, I used 1.6ug DNA in 100ul OptiMEM mix with 4.8ug PEI in 100ul OptiMEM, then mixed them, stand at RT, 20min, then, drop the mixture to the 70-80% confluence cells (containing 1mL of DMEM, 10% FBS, no antibiotics). Then, I changed media after 7h of transfection. After 48h, I observed good signal of GFP signal. However, I found that the confluence of my transfected cell still not increase (still 70-80%), whereas the Control (with PEI but no plasmid DNA) was completely over 100% confluence. I worry that this will affect to my result. Is there any one have experience on this? Is that because of too much DNA using for transfection? Thank you.

Nuoc Non Tran You can try X-tremegene 9 reagent which has close to zero toxicity itself (without pDNA) and transfected HEK293T cells nicely reach confluence even without any change of the media (transfecting CMV-driven fluorescent proteins and others). Typically, I use a "100 ul opti-MEM + 3 ul reagent + 1 ug pDNA" mix, which I add 10 ul (100 ng pDNA) per 500 ul media (e.g. for 24-well plate wells). Though, for HEK293T cells even Lipofectamine 2000 has little toxicity when you change the media a few hours after transfection: cells, again, nicely reach confluence.

Michael Koksharov Thank you... So, you mean that for 12-well plate, 1.6 ug DNA for transfection is too toxic to cell, right? :D

Nuoc Non Tran I am not familiar with the PEI method but assuming it's efficiency is similar to X-tremegen-9 (polymeric reagent) or Lipofectamine-2k (lipofection), this will load 8 times more pDNA into cells (800 ng / 500ul vs 100 ng/ 500 ul), so it could easily be toxic. But only an experiment with titration of different concentrations will tell.

In my hands, when transfecting HEK293T with CMV-driven mVenus using X-tremegene-9 (100 ul Optimem + 3 ul reagent + 'X' ug pDNA--> 10 ul per 500 ul well), the total fluorescence from wells saturates anyway after 1-1.25 ug pDNA per mix.

Strictly speaking, even 100 ng / 500 ul is somewhat toxic but HEK293T cells are sturdy and this is not readily visible: there is a small share of cells that get too much pDNA which can be noticed as being brightly fluorescent and rounded in morphology (vs normally not rounded healthy cells). They don't grow any more but they don't die either for a couple of days post-transfection. Whereas cells that got a more appropriate (less) amount of pDNA (or not at all) still divide fast and reach confluence and high density. The toxicity from getting too much pDNA (the reagent alone is essentially non-toxic) is more evident in U2OS cells which are quite larger and grow slower than HEK293T. Here again the saturation of total fluorescence from the well occurs around 1.25 ug per 100 ul mix, but the growth of cells and ability to attain confluence (and then high density post-confluence) is impaired even below 1 ug pDNA per mix (eventual 100 ng / 500 ul well). In case of U2OS cells the share of cells that got too much pDNA already dies within a day (and can be seen as bright green and visually small spherical cells) but the rest of the cells can't fill the void left by them. In case of U2OS cells the growth (to confluence and dense packing past-confluence) is nearly the same to the reagent alone or to no reagent when 0.5 ug pDNA is used per mix (then very few of these dead over-transfected cells can be visible) but the total fluorescence then is also quite lower.