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

• DOI: 10.1016/j.jim.2008.11.014
• Publication Date: 2008-12-26T09:25:28Z
• Authors: Adriana Tiscornia; Ernesto Cairoli; Maria Marquez; Ana Denicola; Otto Pritsch; Alfonso Cayota
• Keywords: 0303 health sciences; Flow Cytometry; Nitric Oxide; Sensitivity and Specificity; Jurkat Cells; Mice; 03 medical and health sciences; Spectrometry, Fluorescence; Leukocytes, Mononuclear; Animals; Humans; Fluorescein; Fluorescent Dyes

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.