Use of diaminofluoresceins to detect and measure nitric oxide in low level generating human immune cells.
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.
Tiscornia
Adriana
A
Instituto Nacional de Donacion y Trasplante de Celulas, Tejidos y Organos, Hospital de Clinicas, Facultad de Medicina, Montevideo, Uruguay.
Cairoli
Ernesto
E
Marquez
Maria
M
Denicola
Ana
A
Pritsch
Otto
O
Cayota
Alfonso
A
D000818:Animals; D005434:Flow Cytometry; D019793:Fluorescein / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
C111205:4,5-diaminofluorescein; C414689:4,5-diaminofluorescein diacetate; D005456:Fluorescent Dyes; D009569:Nitric Oxide; D019793:Fluorescein