Does anyone has good protocol/reagent for transfecting U937 cells with siRNA? It is a human macrophages and looks like they are quite sensitive and float/die after, even with very low amount of lipid based reagent and oligos on forward transfection.
I am looking to transfect after 3 days PMA differentiation and induced with interferon after siRNA for 2 days.
I believe U937 is a suspension cell line, so electroporation is generally gonna be the way to go. Look for electroporation reagents that are for AML cell lines (like K562: https://altogen.com/product/k562-electroporation-kit-aml-leukemia-cells-ccl243/) and those should be applicable to your transfection. I've noticed that lipofection of suspension cell lines only gets around 10% transfection efficiency, but electroproation can get up to 80% efficiency, especially for siRNA transfection.
hi Ted, thanks for the input, they are monocytes in suspension, but we normally differentiate them into macrophages, meaning that they attach at the time I did the transfection though.
Interesting, I haven't personally conducted such kinds of suspension-turned-adherent cell transfections, but if your cells are adherent then lipofection should work. Given the issues you've been facing, it seems to me on a surface level that your concentration might be too high or that your medium isn't suitable for the cells. Maybe do an optimization round of different concentrations, and that might give a direction to head in. Also you might want to read this study (Article Transfection of adherent murine peritoneal macrophages with ...
) which seems to deal with the same kind of transfection you are.
Hello! our Viromer BLUE transfects U937 cells quite good...
see for example this publication
Article The role of microRNA-5196 in the pathogenesis of systemic sclerosis
They used 75nM of pre-miRNA which shows some optimization compared to the standard protocol (25nM) but nothing special for the workflow of the transfection itself.
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