I know IMR-90, which is a lung fibroblast cell line, has a designated transfection reagent/protocol for it (https://altogen.com/product/imr-90-transfection-reagent-lung-fibroblast-ccl186/). It might be relevant to the experiment at hand. Otherwise, fibroblast reagents (https://altogen.com/product/fibroblast-transfection-reagent-fibroblast-cells/) should work to give good efficiency. It depends a lot on what you are transfecting - silencing with siRNA will generally be more efficient then transfecting plasmids.

We also tried to transfect fibroblasts with the same result. Others succeed using the electroporation INucleofector). But we never tested it yet.

I once cultured the lung fibrablast cells from pig. to my experience, this kind of cells may be not suitable for transfection. because they grow slowly and only could be passaged not more that 10 times with good morphology. my purpose was to find the suitable kind of fibroblast cells to obtain transgenic cloned embryos. if your purpose is a little similar, i will recommend the kidney fibroblast cells. Good luck!

We transfect similar cells mouse embryonic fibroblasts (MEF) by electroporation using BioRad Gene Pulser XCell, 4x10exp6 cells in 400 µl, 320V, 950 µF, 4mm cuvettes in Electroporation medium (10 mM HEPES (ph 7.5), 135 mM KCl, 2 mM MgCl2, 0.2 mM CaCl2, 5 mM EGTA, 25 % FCS.

Maybe it's worth to try this out.

I heard that electroporation is one option. Thank you all for the help! I can borrow the biorad device, so I will try ;)

Thanks a lot!

I have used nucleofector to transfect siRNA oligos into MRC-5 human fibroblast with above 90% success, the efficiency of transfection of constructs depends on quality of DNA but again it is in the range 50 -60% even more for pMaxGFP vector provided by Lonza as a control. Details of protocol could be sent upon request.

Electroporation uses a lot of DNA. We use a kit called Transit 2020 from Mirus. It has worked well for primary fibroblasts which were very very hard to transfect. Perhaps you could ask them for a sample and see whether it works. If you do decide to go with electroporation, a bit of DMSO in your transfection media increases electroporation efficiency. Good luck!

Aditi thanks a lot, I've never heard of transit... is it too toxic for the cells? It seems that I have to try it.

It is not very toxic.

Here is the site: http://www.mirusbio.com/products/transfection/transit_2020_transfection_reagent

They do give out samples.

Thanks ;)

Perhaps an older method works?

- Mooibroek H, de Jong B, Venema G. Repair of UV damage in plasmid DNA by human fibroblasts. Mol Gen Genet. 1984;195(1-2):175-9.

- Hans Mooibroek, Annika C. Arnberg, Bauke Jong and Gerard Venema. Effect of concentration on the subsequent fate of plasmid DNA in human fibroblasts. Molecular and General Genetics MGG. Volume 199, Number 1 (1985), 82-88, DOI: 10.1007/BF00327514

For murine cells an easy method with an amazing transfection efficiency is using a retroviral system, see link of the Stanford University with protocols and tutorials: http://www.stanford.edu/group/nolan/retroviral_systems/retsys.html.

We also had trouble transfecting primary murine lung fibroblasts using different otherwise efficient lipofection reagents. However, we achieved very good efficiency using retroviral transfection with the Plat-E system combined with pMSCV or pBABE vectors. Since these vectors are derived from murine viruses, they are safety level 1 and can thus be used in most labs.

Hi! I am having a similar experience. Would like to know a follow up on anything that worked in transfecting NHLF from Lonza. Thanks!

I know IMR-90, which is a lung fibroblast cell line, has a designated transfection reagent/protocol for it (https://altogen.com/product/imr-90-transfection-reagent-lung-fibroblast-ccl186/). It might be relevant to the experiment at hand. Otherwise, fibroblast reagents (https://altogen.com/product/fibroblast-transfection-reagent-fibroblast-cells/) should work to give good efficiency. It depends a lot on what you are transfecting - silencing with siRNA will generally be more efficient then transfecting plasmids.