Hi Liuyuan, MDA-MB-231 is truely hard to transfect, one choice is to use lentivirus or adenovirus, their infection efficiency is much better.

Genemedi launched a product of Lipogene, comparable to lipofectamine 3000, but with fewer cytotoxic effect. You could find more information on this website: www.genemedi.net/i/lipogene-transfection-reagent

Hope it helps.

We also used MDA-MB-231 cell line in our experiments, the transfection efficiency is very high, all most 85%, using the normal transfection protol with lipofectamin2000. My suggestion is that you need to check the cell line first, maybe it is not MDA-MB-231.

It's possible that the cell line changed after long time culture, and you could get a new cell line from other labs, or you could buy one from Xiehe cell line base. It's quite cheap, you could try...

I did transfection experiments on MDAMB231 and MCF7 using Fugene (that is quite similar to Lipofactamine) and the efficiency was not bad (50-60%).

Have you checked your cells for mycoplasms contamination? Have you tried to clean up your plasmid?

I use to transfect my MCF7 cells with Turbofect. It worked pretty well in my hands so far (~80%). I have no experience with MDA-MB-231, but I would suggest you give it a try, since Turbofect is working well on all cell lines I tested and since it is pretty cheap compared to FugenHD or Lipofectamine. Another alternativ might be to use PEI (Polyethylenimin) based transfedtion protocols.

As Manuela already suggested I would think about using a good preparation of plasmid.

Good Luck,

Kai

P.S.: I case you need a protocol for PEI just contact me.

I've got to ask you some questions that may seem pretty stupid but at same time they can help (somehow).

Are you using free-serum (and free-antibiotic) medium to perform your transfections?

I'm asking this because both serum and antibiotics decrease the efficiency of transfections.

Another thing is: how confluent your cells are... my recollection is that this cell line requires at least 80% (I know most of cell lines also requires this %, but for some cells this is more critical!)

plus, To change the medium, after 3-4h of transfection, replacing with a full medium is very important. That means cells will be kept during transfection with a very poor medium (serum free)... after this period (3-4h) I usually change the medium, you know...

Finally I also agree with our colleagues wrote here, about your cell line... I think it's worthy to check them.

best wishes,

@lex

HI Liu

We have transfected MCF7s and MDA-MB-231's many times and have observed transfection efficiencies (around 50-60%). Most modern day transfection reagents will transfect most commercially available, commonly used cell lines to a similar(ish) efficiency.

It is clear from our data (and from that posted above..and below) that 231's can be efficiently transfected. I would start to troubleshoot your transfection reagent/cells/culture conditions/transfection assessment method first. With regards to transfection reagent, Lipo2000/FuGene/etc will all be able to transfect these cells. As Kai mentioned, I would strongly suggest moving to jetPEI as in my opinion it has largely superceded those reagents mentioned above. Regarding your 231 cells, I'd try another vial, check your 231's are actually 231's (eg by STR genotyping), or buy another batch.

It sound to me like it's a technical issue with regard to the transfection. Have you transfected this prep of DNA before (ie, is it dirty)? Perhaps it's worth cleaning up the DNA and having another go.

Good luck

Al

Thanks for all your suggestions to my problem. After consideration it might be the cell line makes the transfection efficiency very low. To transfection reagent and plasmid, we check it in different cell lines and the results present well. Our MDA-MB-231 cell line looks ok in shape and condition though we didn't know how much passages it was. we decided to buy a new cell line at xiehe cell line resource centre after spring festival and try to repeat this experiment again. In addtion we have tried lot's of transfection reagents includ Polyplus/FUGENE ect. finnally only lipofectamin2000 can make about 1% transfection efficiency.

Thanks Fankai Ji and Alasdair Russell give me MDA-MB-231 cell line's transfection efficiency in your experiment.

Yuanwu Liu

I changed the MDA-MB-231 cell line from xiehe cell resource centre the transfection efficiency reached nearly 90% with lipofectamin2000. thanks for all your help!

Dear Liu,

Would you mind sharing your Lipofectamine 2000 protocol for the MB-231 cell line?

E.g. how much Lipofectamine to how many ng of plasmid? I have a fresh batch of cells but the highest efficiency I can reach is only 50+%

Thank you very much!

Dear Muhd

Lipofectamin 2000 might be not efficient to transfer this cell line. After compared different transfection reagent I recommend you use polyplus to transfer this cell line. my 90% transfection efficiency just account to my control plasmid. my experiment construct nearly 8kb and the transfection efficiency reduced to 20-30% or so. Hopes this information can help you.

Thank you!

Dear Olivia,

Thanks for your reply, to MCF7 cell line is fine to transfection. after reading references MDA-MB-231 cell  line is hard to transfection there are no perfect solutions in this problem.

Best,

Yuanwu Liu

MDA-MB-231 can be unresponsive to generic transfection reagents, and given that the cell line is significantly different than MCF-7, it isn't that surprising that you're getting different results. MDA-MB-231 belongs in the MDA-MB family, so directed transfection reagents (https://altogen.com/product/mda-mb-transfection-reagent-breast-adenocarcinoma/) should work for all of the cell line members. 1% efficiency is not acceptable, so you'll need to look at established protocols and follow them to get better results.

We have transfected MDA-MB-231' and RAW cells with lipofectamin LTX. and its the best reagent for hard cells to transfect.

For GFP protein transient transfection in MDA-MB-231, would you suggest lipofectamine or PEI? Any protocol available? Thank you in advance

Hi Liuyuan, MDA-MB-231 is truely hard to transfect, one choice is to use lentivirus or adenovirus, their infection efficiency is much better.

Genemedi launched a product of Lipogene, comparable to lipofectamine 3000, but with fewer cytotoxic effect. You could find more information on this website: www.genemedi.net/i/lipogene-transfection-reagent

Hope it helps.