I need to transfect U251 and I've been using Lipofectamine 2000 with poor efficiency (less than 30%). Can anyone provide me a good transfection protocol for this cell line?
EZ Biosystems has U251 specific transfection reagent. Here is the link.
http://ezbiosystems.com/view2.asp?d_id=315&Name=U251%20Cell%20Avalanche%26%23174%3B%20Transfection%20Reagent%20(human%20glioblastoma%20cell)
Hello. A few other things to consider for transfection before switching the reagent are the cells themselves, the purity of the DNA, and buffers used (such as OptiMEM). Health of the cells, proper passaging, media and cell density at the time of transfection can affect efficiency by almost 5-10%.
Something else to try, if you are looking for high efficiency transfection, would be to try transfecting mRNA, which does not require nuclear entry, and provides >90% efficiency in difficult to transfect cells and even primary cells (like neurons and hepatocytes).
Unfortunately, we haven't tested this cell line with the new Lipofectamine 3000, but I've listed some optimization steps that you can use to help improve transfection with DNA or RNA, for a wide variety of cell types, even the difficult to transfect ones:
• Performing the recommended reagent protocol, with the indicated doses, will help to determine the “sweet spot” of the reagent for a particular cell type
• Optimizing for cell density so that the cells are 70-90% confluent on the day of transfection, can help to improve efficiency by almost 10-15%
• Maintaining healthy cells in log phase during sub-culturing
• Controlling cell passage number post thawing is important for the health of the cells; most cell types should be used between 4 and 25 passages for optimal transfection
• Using the recommended cell culture media
Hope this helps!
https://www.lifetechnologies.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html
https://www.lifetechnologies.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-messengermax.html
If you know of anyone in your institute or building with an Amaxa nucleofector, see if you can borrow it. You'd need to buy a kit for your cell type, but I found it great for hard-to-transfect cells that don't work with lipofectamine.
Hi Wanda,
I can recommend DreamFect Gold for your application
http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html
or another another option would be to use Magnetofection that is dedicated to hard to transfect cells, especially a kit called Magnetofectamine that combine magnetic nanoparticles with lipofectamine 2000, you can find infos here
http://www.ozbiosciences.com/transfection-dna/49-magnetofectamine-magnetofection-lipofectamine2000-combimag.html
If you want sample let us know: [email protected]
Good Luck
Olivier
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