Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cellsin vitro

Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells invitro . The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 �M) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikertet al. ,FEBS Lett 2001, 506:131–134) on aspects of validation procedures as well as limitations and pitfalls of this method.