I have ordered lipofectamine 2000 from Thermo Fishers Scientific. The product has been provided with a protocol, but does not clearly reports the incubation times. I have tried transfection as per protocol, but it seems the cells had toxic effect on them post- transfection. They had clumped to form aggregrations. Most of them were swollen and reduced in size.
It's likely that you will get cytotoxicity from a generic reagent; each cell line has its own quirks, and finding the right optimal conditions for transfection generally requires some time. Here's a HEK293 transfection reagent (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/) that is optimized for the cells, and so you shouldn't be getting cytotoxic results. Make sure that you're running control samples with just the transfection reagent to see what percentage of cells lose viability, and that will help you calculate efficiency more accurately.
Rupa,
Lipofectamine 2000 is cationic based lipid delivery system, which transports DNA into cells by fusing with plasma membrane. HEK-293 is easily transfectable cell line. I attached the protocol which we use for Invitrogen L-2000.
If your cells are dying post-transfection, you have to optimize the following,
- how long you keep the transfection mixture, DNA plus lipofectamine 2000.
- how much lipofectamine and DNA, this depends on whether you use 12 or 6 well plates or larger dishes and the cell density.
L2000 is quite toxic. You might want to consider very old, yet effective transfection of HEKs with calcium phosphate. Straightforward, cheap, efficient, non-toxic
Thank you all for your replies. I'll try out the transfection and will let you know which one worked.
Hek293 are basically easy to transfect but toxicity can be an issue with some well known reagents.
Try ScreenFect A which is more gentle to the cells together with high efficiency transfection performance.
As we do routinely transfections with Hek293 we do have an optimized protocol for them.
We can provide you with a free sample for testing.
http://www.incella.com/products/screenfect%c2%aea/
Rupa,
Apart from using SFAF medium, you can use Serum containing antibiotic free medium for incubating DNA-L2000 complex. (Some cell lines are sensitive to repeated DPBS washes or serum starvation which makes them detach(not dead) readily. For them, it is preferred to seed them in Antibiotic free medium. Next day, add Complex to the plate and incubate fro 4-6hr, then replace with complete medium after giving DPBS washes. If you are confident enough you can maintain cells in antibiotic free conditions.)
But for complex preparation we have to use SFAFmedium or Opti-mem serum free medium.
All the best.
It's likely that you will get cytotoxicity from a generic reagent; each cell line has its own quirks, and finding the right optimal conditions for transfection generally requires some time. Here's a HEK293 transfection reagent (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/) that is optimized for the cells, and so you shouldn't be getting cytotoxic results. Make sure that you're running control samples with just the transfection reagent to see what percentage of cells lose viability, and that will help you calculate efficiency more accurately.
- Badges
- Science method