Hello Brett Seese

If you dilute culture supernatant with culture medium, you should also dilute the standards in the same matrix as your samples otherwise, as you mentioned your results will be skewed if your samples and the standards are not in the same matrix.

Best.

In my experience when my samples have too high of a concentration of my protein of interest, I dilute my samples, which are the cell supernants, 1:10 in the reagent buffer (also the block, usually PSA-BSA). If you were to dilute the samples in the cell culture medium, I would think it would have to be after the transfection took place, as you would not want to throw off the ratios of your transfections. If that is the case, the protein content should still be the same regardless which matrix you dilute your samples in. As for the standards, the manufacturer usually tells you what to dilute your standards in the protocol, so I would refer to that for what to use. I have done sandwich ELISAs with the standard diluted in PSA-BSA and my samples are from bacterial-infected mammalian cells supernants and my results have been reliable.

To dilute the samples always use the buffer which you use to prepare your standards. For examples, if your using buffer X to prepare serial dilution of your standard, use the same buffer X for dilution of your cell culture supernatant. Not the cell culture media