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J Immunol Methods. 2009 Mar 15;342(1-2):49-57. doi: 10.1016/j.jim.2008.11.014. Epub 2008 Dec 25.

Use of diaminofluoresceins to detect and measure nitric oxide in low level generating human immune cells.

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Instituto Nacional de Donacion y Trasplante de Celulas, Tejidos y Organos, Hospital de Clinicas, Facultad de Medicina, Montevideo, Uruguay.


Nitric oxide ((*)NO) has been implicated in multiple physiological and pathological immune processes. Different methods have been developed to detect and quantify (*)NO, where one of the principal difficulties are the accurately detection in cellular system with low levels of (*)NO production. The choice of the (*)NO detection method to be used depends on the characteristics of the experimental system and the levels of (*)NO production which depend on either the organism source of samples or the experimental conditions. Recently, high sensitive methods to detect and image (*)NO have been reported using 4,5-diaminofluorescein-based fluorescent probes (DAF) and its derivate 4,5-diaminofluorescein diacetate (DAF-2 DA). This work was aimed to adapt and optimize the use of DAF probes to detect and quantify the (*)NO production in systems of high, moderate and low out-put production, especially in human PBMC and their subpopulations. Here, we report an original experimental design which is useful to detect and estimate (*)NO fluxes in human PBMC and their subpopulations with high specificity and sensitivity.

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