I want to transfect THP-1 cells (and if preliminary experiments work, I plan to later transduce them with a virus to generate a stable cell line). For my functional experiments, I want them to be differentiated into adherent macrophages using PMA.
Ideally, I would like to transfect the adherent cells, for sakes of the time-course of knock-down.
I understand that THP-1 suspension cells are very difficult to transfect using lipid-based reagents, but nucleofection works well. I would like to know if once they are PMA-treated, whether the cells will be more amenable for transfection with lipid-based reagents. I don't really want to differentiate the cells, get them back into suspension for nucleofection (trypsin or EDTA?), and then re-plate. Any suggestions?
My experience is that THP-1 cells are not very transfectable even when they are differentiated. I usually transduce the pre-differentiated THP-1 cells with a retroviral or lentiviral vector and then differentiate the cells following selection. This works well to both over-express a gene of interest and to knock-down a gene.
Ok, thank you very much for your answer. I was hoping in a pilot experiment to just do transient transfections, as I don't have any experience in packaging vectors in 293T cells for the stable transductions. But looks like I will have to jump straight in at the deep end!
I have tried siRNA transfection to adherent, differentiated THP1 using siLentFect from BioRAD. It works well from my experiences.
I have just tried to electroporate THP1 and induce the differentiation right away after electroporation. According to the manufacturing procedure, this should work. I will let you know if this works well from my side.
Thank you Wanida, that would be great. My only concern with that approach was that you would need the time needed for knockdown to coincide with the time needed for differentiation, i.e. you don't want maximum knockdown to occur before you achieve differentiation. But I think it could still be the best approach and I will be very interested to hear your results!
I see. I am actually working on the electroporation to overexpress my genes in those cells. For KD, I used siLentFect transfected directly on differentiated macrophage. The cells seems fine at 24-48hrs post siRNA transfection. However, it depends on your final assay, the cell health can be affected. My KD efficiency was very good (
Unfortunately, my electroporation then differentiation cells did not yield overexpress THP1 macrophage.... I am trying some other methods currently. I hope you have some luck with your experiments.
oh, sorry to hear that. Perhaps the differentiation process affects the overexpression of the genes. Good luck. I have yet to try my KD....
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