I have been doing transfection of THP-1 cells, U937 cells and J774A.1 cells with NLPR3, ASC and Caspase 1 containing vectors together using Lipofectamine LTX reagent but I am not able to find overexpression of these proteins. Infact I tried to transfect a GFP containing vector but no green fluorescence is observed.
To validate my Lipofectamine LTX reagent, I have transfected HEK293T cells using GFP as well as NLRP3, ASC and Caspase 1 containing vectors and I found overexpression of these proteins. This proves my transfection reagent is working fine.
But I need to transfect in immune cells for my experiments. How to achieve this co-transfections of three different plasmids containing NLRP3, ASC and Caspase 1? These plasmids have c-Myc tags and CMV promoters.
Please help!!
Hi! Unfortunately plasmid transfection is rarely succesful in immune cells due to recognition of dsDNA as foreign material (innate immunity). If you need only transient expression, you may think about using mRNA instead...
for more details, https://viromer-transfection.com/mrna-transfection
Best, Sandra
Thanks a lot Sandra and Jhanvi for your suggestions. Since pDNA is hard to transfect in immune cells (especially the suspensions cells like THP-1, U937 cells), I will try to do reverse transfection protocol using Lipofectamine LTX reagent.
Will let you know my this weekend.
Good luck!
Difficult but not impossible... just a question of optimization efforts. With our Virmer RED we manage transfecting plasmid DNA in THP-1 at around 10-25%, J774 around 10% and around 20% for U937. But again keep in mind that with mRNA, you can reach 40-50%, 70% and 40% respectively...
I did reverse transfection of U937 cells with GFP expressing plasmid and I got the over-expression of GFP. I have attached an image of my result for the same at different time duration.
Now I will be transfecting this U937 cells with my desired plasmids and lets see what I get. Thanks Jhanvi for your kind suggestion.
Gundappa Saha Thanks for updating your results. I am at the same point as you were in June 4, 2018 starting out transfection experiments in U937 cells. Have you perfected the technique? I am concerned about the number of cells as my analyte detection varies with the cell density. Since I also have lipofectamine reagent, it would be good for me if you could share the protocol. Regards.
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