Detection and Imaging of Nitric Oxide with Novel Fluorescent Indicators:  Diaminofluoresceins

Abstract

Nitric oxide is a gaseous, free radical which plays a role as an intracellular second messenger and a diffusable intercellular messenger. To obtain direct evidence for NO functions in vivo, we have designed and synthesized diaminofluoresceins (DAFs) as novel fluorescent indicators for NO. The fluorescent chemical transformation of DAFs is based on the reactivity of the aromatic vicinal diamines with NO in the presence of dioxygen. The N-nitrosation of DAFs, yielding the highly green-fluorescent triazole form, offers the advantages of specificity, sensitivity, and a simple protocol for the direct detection of NO (detection limit 5 nM). The fluorescence quantum efficiencies are increased more than 100 times after the transformation of DAFs by NO. Fluorescence detection with visible light excitation and high sensitivity enabled the practical assay of NO production in living cells. Membrane-permeable DAF-2 diacetate (DAF-2 DA) can be used for real-time bioimaging of NO with fine temporal and spatial resolution. The dye was loaded into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner.

Amyloid β induces neuronal cell death through ROS-mediated ASK1 activation

“…We next examined whether Ab induces NO within cells by using a fluorescence probe, DAF-2 DA, that can directly detect NO. 29 Although NO was easily detected after treatment with NOR1 (Figure 3c), we could not detect apparent production of NO by treatment with Ab (Figure 3d). These results suggest that NOS-induced synthesis of NO may not be the main mechanism for Ab-induced ASK1 activation.…”

“…Ab [25][26][27][28][29][30][31][32][33][34][35] and Ab 1-42 were purchased from Bachem and American Peptide Company, respectively. Ab [25][26][27][28][29][30][31][32][33][34][35] was dissolved in water at 1 mM and Ab was dissolved in water containing 0.1% NH 3 at 1 mM.…”

“…Ab [25][26][27][28][29][30][31][32][33][34][35] was dissolved in water at 1 mM and Ab was dissolved in water containing 0.1% NH 3 at 1 mM. Dissolved Ab peptides were mixed with the same volume of PBS and incubated at 371C for 3-5 days before use.…”

Mechanisms of β3‐adrenoceptor‐induced eNOS activation in right atrial and left ventricular human myocardium

“…Diaminofluorescein (DAF-FM DA) is converted via an NOspecific mechanism to an intensely fluorescent triazole derivative (Kojima et al, 1998). We have used in the present study DAF-FM DA to detect changes of the NO level induced by BRL.…”

In vitro and in vivo defensive effect of probiotic LAB against Pseudomonas aeruginosa using Caenorhabditis elegans model

“…A harmonized population of L1 arrested worms were then placed on NGM agar plates seeded with LAB. Five stage [L1 – after egg 5 stage of egg development and immediately after hatching Nervous, Reproductive and coelomocyte system developmental stage; L2- few cell division after L1 stage leads to possess developed reproductive and nervous system (L2 stage also known as dauer larva; L3- posse matured reproductive system; L4- young adult posse fully developed reproductive system and Adult- hence completing its 3-day reproductive life cycle] [29]. In that L4 nematode were picked manually and moved to a new plate.…”