I´m using 1:6 DNA:lipofectamine ratio. 24 hrs after transfection the cells looks in bad conditions and just few cells are transfected (GFP expression). I will be glad to have suggestions.
Cytotoxicity from transfection reagents is a common problem. Unless you find reference to transfection of these specific cells, I'm afraid it's a bit of a trial and error affair.
Start with the reagent you have and follow the protocols for varying the DNA:reagent ratios and cell confluence. You can also ease the cytotoxicity by washing the reagent off the cells after 5 or 6 hours. This will not increase the efficiency though. If you can, also try different reagents - I find FuGene6 works well for some cell lines that get sick in lipofectamine, for example.
If you have access to the equipment then electroporation is also something to try.
Good luck!
I do not have any experience with this cell type specifically, but I have transfected other cell lines using electroporation method. You can achieve upto 80% transfection efficiency by electroporation. As already suggested you have to standardize the conditions for transfection. You can-
1. Use various concentration of good quality supercoiled DNA to check which conc. gives you maximum transfection efficiency.
2. Use higher cell number. Make sure your cells are healthy before you transfect. Split them the previous day of transfection.
3. Use warm and fresh media for transfection purpose. You need to take extra care for your transfected cells to help them cope up with the foreign DNA.
4. Change media soon after your transfected cells are attached (6-8 hours after transfection, depending on cell type). Then again after every 12 hours (for delicate cell lines) and check efficiency after 36-48 hours.
Hope it helps!
Dear Eric,
when transfecting cells, toxicity may come from several sources, and the inherant toxicity of the transfection reagent may be a big part. Moreover, non-adapted transfection conditions greatly influence the becoming of cells.
In order to try reducing toxicity while enhancing efficiency I will suggest you two solutions:
- to use Magnetofection*** in association with Lipofectamine. CombiMag is a formulation of magnetic nanoparticles that was designed to be combined to any transfection reagent and its efficiency was proven in multiple articles (you can refer to our citation database in the link below)
- to use an alternative, all our transfection reagents are biodegradables and would be of great interest as an alternative as demonstrated by Orth P. et al (Mol Biotech 2008).
I will insist on the 1st alternative. Actually using CombiMag associated to your transfection system would allow reducing the amount of DNA used, reducing the volume of transfection reagent use and thus lowering the toxicity while gaining significant improve in transfection efficiency.
It is important to note that the magnetic nanoparticles will not create some hole into the cell membrane, the magnetic field attracts and induces clusters of magnetized complexes that enter through classic endocytosis pathways. In this way the cell membrane remains intact preserving cell from suffering.
I hope this would be of help for you,
please do not hesitate to contact me via researchgate or directly at: [email protected]
Cedric
MAGNETOFECTION
*** The Magnetofection technology uses a magnetic field to attract and concentrate complexes of magnetic nanoparticles and nucleic acid onto the cell surface as demonstrated by Grześkowiak BF et al, Pharm Res. 2014 Jul 18. (http://www.ncbi.nlm.nih.gov/pubmed/25033763).
http://www.ozbiosciences.com/module/citationfinder/default
Hello everyone.
I don´t have access to an electroporation equipment so, I´m testing different ratio DNA:Lipofectamine to determine the best conditions.
Thanks for the help.
Hi Eric,
I can see this is an old thread, but I'm having the same problem transfecting Saos-2 cells with Lipofectamine2000, and I would like to know if you could determine suitable conditions to do so.
Thanks in advance.
Hi Ayax,
Well I don't know what was happen, I just change the batch of Lipofectamine2000 and now it is working very well.
The strange thing is that the Lipofectamine2000 with troubles to transfect Saos2 is working perfectly for other cell lines.
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