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As indicated in the insert, as long as the standard curve is titrated in the same culture media that the secreted cAMP/cGMP is in; then, any small changes that occur in optical density (OD) should be comparable across the plate keeping your samples and standards. Phenol Red-containing RPMI1640 and DMEM+Glutamax spiked with cAMP/cGMP were used when developing these kits and a slight increase in OD was obtained but this was reflected in the samples as well. Enzo Life Sciences does advise end users to optimize their own sample dilution and try their own spike/recovery test in their samples to ensure good recovery and linear data.