Hi Seema, I assume you are doing overexpression transfection? How are you measuring the efficiency? Also how long are you leaving the cells post transfection before you measure? I had to leave mine until 48 hours for maximal efficiency but I still detected protein after 24 hours. For overexpressions I have been told 30-40% is enough to see an effect. The other thing you can do is: if your cells are adherant transfect them after trypsinisation and resuspension, or try varying the cell confluency. I have tried lipofectamin LTX too but I found it was very toxic for my cells but I know others here who have used it with no problem.

Hi Seema. i do agree with Sara that you should not measure your efficiency before 48 hours. and from my experience, transfecting the cells directly after trypsinizing them gives excellent results. and for that i use 60-70% cell confluency at transfection time depending on how fast does your cells grow.

Hi Seema,

I have been working with A549 for quite a bit and I found Lipofectamine 2000 or LTX to be the best transfection agents. I have used it both for stable and transient transfections. However, Lipofectamine 2000or LTX require cells to be 70-80% confluent at the time of transfection. Otherwise you will have a lot of cell death. I have tried Fugene HD as well but it gives a low transfection efficiency (similar to what you mentioned) even after 72h. So, I would suggest you use Lipofectamine 2000 or LTX and optimize cell confluency for best results.

Thank you so much.I am trying for stable transfection and looking for long expression measure.I measure them by GFP and FACS.

have you got toxicity or cell death by trypsinizing and transfecting immediately.

I think lipofection2000 can increase your transfection efficiency. I have used FugeneHD for transfection,but the transfection efficiency is somewhat low.

One can even perform subsequent transfections of cells to further increase transfection efficiency. e.g. perform simultaneous plating/transfection on day0 (as already mentioned by Randa, immediate transfection of newly trypsinised - or even better Accutase detached - cells can be more efficient and saves one day) then change media and perform another transfection of the now adherent cells and then change media 4-5 hours after the 2nd transfection and leave overnight.

We (a chemistry and Biology lab collaboration) have developed a new type of transfection reagent here at Karlsruhe Institute of Technology (KIT, Germany) called ScreenFect that shows improved efficiency compared to Lipofactamine 2000 or LTX for many cell lines. It also has the advantage that, generally, less plasmid DNA is required.

Contact Incella (www.Incella.com) to ask for a free sample to test and use the optimisation protocol. This is a new spin-off company that is commercializing ScreenFect.

Which viability you have to this efficiency? Effciency alone is not a criteria if viability is low.

viability is good about 60-70% .Effeciency is only coming low .but now i have tried mirus transit pro reagent and this gives upto 90% effeciency for a549 but about 50 % cell death .

I did transfection recently, it would yield a higher transfection rate and low cell death if you have 80-90% confluent cells

Hi there, I wander if you solved the efficiency problem and how. Can you share your progress with us? Although the topic is old now, I wander if you ever checked your Mycoplasma status of contamination of your cells... 

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