These are normal human fibroblasts derived from foreskin. We ordered them from ATCC. I tried transfecting them with a generic GFP expressing vector using both Turbofect and Transit X2 and got nothing. I will try nucleofection this week but was hoping to use a simpler method. Suggestions from anyone who has worked with these cells are very much appreciated.
BJ is difficult to transfect, but for siRNA transfection with some reagents (https://altogen.com/product/bj-transfection-reagent-skin-fibroblast-cells/) you can get up to 80% efficiency. There are of course several factors affecting the overall efficiency, like the confluency of cells, the medium, and the size of the cargo you are transfecting, but generally speaking following protocols should give you decent results.
Dear Dominica,
Fibroblasts can be considered hard-to-transfect cells and it's quite true that chemicals and classic transfection reagents show some limits.
That's why I would highly recommend you to try Magnetofection method that uses a Magnetic field to concentrate complexes of magnetic nanoparticles/nucleic acids onto the cell surface.
Even if it is considered as a physical way of transfecting cells, the Magnetofection uses the classic pathways to transfect cells (endocytosis...); no hole is created during the transfection process and cellular integrity remains intact.
All the material are biodegradable and compatible with cell and organisms life.
This methods is really efficient for primary cells and hard-to-transfect cells ; the best example is be the transfection of primary neurons, primary endothelial cells, primary amniotic cells...
Moreover, there are some references using Magnetofection to transfect fibroblasts. For more information you can have a look at :
Frolov A., (J Biol Chem. 2013) : synovial fibroblasts transduced by magnetofection, Ji JH., (J Oral Pathol Med. 2009) : mouth epidermal fibroblasts transfected with siRNA by magnetofection or Couchoux H., (J Physiol. 2007) : fibroblasts from foetal fibers. the links are the following ones :
http://www.ncbi.nlm.nih.gov/pubmed/23814065
http://www.ncbi.nlm.nih.gov/pubmed/19453842
http://www.ncbi.nlm.nih.gov/pubmed/17317753
I would be very glad in assisting you trying the Magnetofection technology, so please do not hesitate to contact me either via Researchgate or directly at [email protected]
good luck with your experiments,
have a nice day,
Cedric
Thank you Cedric. I would like to try this reagent. We have had some success with various reagents but it is limited and only works for very basic plasmids such as one expressing a cytosolic fluorophore. More complex and larger plasmids transfect very poorly.
I've looked at the range of magneto reagents and have decided to try the PolyMag neo. What do you recommend? Also, where do we get the magnetic plate?
Thanks again for your suggestion, much appreciated.
Mina
Dear Domina,
PolyMag Neo would definitively be the candidate ; you can contact me directly at [email protected] so that I will forward your request to our commercial department: they will take care of sending you some samples and how to get a magnetic plate.
looking forward earing from you soon,
Cedric
Hey Domina,
Can I ask you how the magnetofection turned out with these cells? More specifically:
1) Did you get a better transfection efficiency with the generic GFP expressing vector?
2) What was your transfection material of interest?
3) Did you eventually try the nucleofection, and if so, which program worked best for you?
Kind regards,
Shana
BJ is difficult to transfect, but for siRNA transfection with some reagents (https://altogen.com/product/bj-transfection-reagent-skin-fibroblast-cells/) you can get up to 80% efficiency. There are of course several factors affecting the overall efficiency, like the confluency of cells, the medium, and the size of the cargo you are transfecting, but generally speaking following protocols should give you decent results.
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