I am growing adherent primary epithelial cells in supplemented DMEM/F12 and testing the supernatant for inflammatory cytokines. I purchased a commercial sandwich ELISA to test for analyte. Unfortunately the DMEM/F12 media I grow my cells in has an extremely high background potentially clouding any significant results. I know its the media because I ran some wells with just the media vs media with FBS vs media with bovine pituitary extract and the plain media well showed just as much background as the supplemented media. Has anyone else experienced this and if so did they find a way to remedy it? Does using RPMI instead of DMEM/F12 give a lower background??
Whitout exactly knowing which kind of assay you're using, I'd try to use medium without phenol red, if you're not doing already. Phenol red containing medium can easily interfere with colorimetric assays. DMEM/F12 exists without phenol red, so it's worth a try. If your reradout will be a flourescent signal, you can try FluoroBrite DMEM Medium, which was designed to enhance the signal-to-noise ratio of fluorophores and it exhibits a low background fluorescence.
Dear check the kit you are using if it is good and ensure you adhere strictly to the manufacturer's instructions.
Sounds like you could benefit from either Dialyzed or charcoal-stripped FBS.
Best of luck!
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