Hello,
I have been trying to transfect Cancer Associated Fibroblasts (CAFs) with a plasmid of interest. I have tried Lipofectamine, Lipofectamine 3000, Fugene HD, and TransitX2. All of these reagents show very less transfection efficiency. I have cross-checked it with a control plasmid and obtained same results.TransitX2 and Lipo 3000 are also having high toxicity.
I was wondering if anyone has used any particular reagent for transfecting CAFs with good efficiency? Alternatively, are there any good reagents designed for primary cells that I can use.
Thanks!
Hello,
We are currently using TransIT-LTI (https://www.mirusbio.com/products/transfection/transit-lt1-transfection-reagent) as a transfection reagent for our murine and primary cell lines and it has been working quite well.
Could you share the protocol you use to set up your transfection/DNA mixes? It may not necessarily be the transfection reagent that is the only factor in poor transduction efficiency.
Best,
Areya
Hello Areya,
Thanks for the feedback.
For my transfection I use about 80% confluent cells grown in P/S free medium.
I prepare my DNA mix according to the protocol. I use 1ug DNA in 24-well plate. If using Fugene HD, I use DNA:Reagent ratio of 1:3. The transfection mix is prepared in Opti-MEM, incubated in room temperature for 10 mins and added drop wise to cells. I change medium after 8hrs.
The CAFs I am using are isolated from patients but immortalized.
Best,
Argha
Hi Areya,
I have used Viafect transfection reagent and worked very well with many different cell lines. Here is the link.
https://worldwide.promega.com/products/luciferase-assays/transfection-reagents/viafect-transfection-reagent/?catNum=E4981
Hello Argha Ghosh
In my hands, plasmid transfections for CAFs (both primary and hTERT ones) are not the best. I would advise you to move in this sense to Lentiviral infection.
For transfection per se (siRNA) for primary cells (CAFs, normal fibroblasts and normal epithelial cells) Hiperfect transfection work very well, with low toxicity and high efficiency (In case interested, one key note would be decreasing the Hiperfect volume suggested by them by 20%).
Good Luck
Hi Lina,
Thanks so much for your feedback. I have been considering doing a lentiviral transduction as opposed to plasmid construct. The plasmid transfection was mainly as a quick pilot experiment to see if my plasmid works in CAFs.
I will consider your suggestions!
Best,
Argha
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