I agree with Shyam that your concentration is a bit off - try following the advice given above. One thing to add though - sometimes with liposome-based transfection the cargo can be a bit too big and physically be resistant to entering a cell. Try using a complex condenser (like the one in the kit below) and see if you can achieve better results.
https://altogen.com/product/a549-transfection-reagent-lung-carcinoma-ccl-185/
Have you tried recovery step in your transfection protocol? I used high transfection setting in the machine and added recovery step in the protocol. It's just a step of incubating your transfection tubes (immediately after transfection) in the CO2 incubator before you transfer the cells to culture plates. This should help to increase cell survival.
I think A549 cells are not very difficult to transfect. One can get over 40-50% transfection efficiency with plasmid DNA and over 85% with siRNA. What is your transfection protocol? Try Lipofectamine 2000 at 1:4 ratio (plasmid ug vs lipid), cells should be around 80% confluency at the time of transfection to reduce toxicity. Make sure your cells are not contaminated (mycoplasma etc) before you start. Hope this information is helpful.
Like Shyam, I do not agree to classify A549 into the hard-to-transfect cell list.
Our customers reach usually between 50 and 80% of efficiency for plasmid transfection with Viromer RED, and around 60-70% of silencing through siRNA transfection with Viromer BLUE.
Please, let me know if you want to test our products.
Bests,
Sandra
Wang Xin: I think your ratio of DNA:lipid is way off . 1:8 is too high, this might be the reason for low transfection efficiency. On the side not, what fluorescent protein were you looking at? GFP?? The normal tissue culture coated plates are very thick and if you get low expression of fluorescent proteins in the CFP/GFP wavelength, they might be bit difficult to detect.
Kamlesh Ganesh Pawar
Sorry how much time it needed for the transfection tubes to be incubated in the CO2 INCUBATOR before adding them to the culture plate ?
Tranfection mixes should me made using pre-warmed OptiMem media (37C) but the transfection mix should be allowed to sit at at room-temperature for 20 (plasmids) or 30 (siRNA) minutes.
- Badges
- Science method