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

Tiscornia, Adriana; Cairoli, Ernesto; Márquez, Marı́a Gabriela; Denicola, Ana; Pritsch, Otto; Cayota, Alfonso

doi:10.1016/j.jim.2008.11.014

Cited by 19 publications

(21 citation statements)

References 47 publications

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“…1A); accordingly, 40 lM was selected. Importantly, this concentration is at least fourfold higher than the concentration required to measure levels of intracellular NO in mammalian cells which ranged from 0.1 to 10 lM (8,28,29,35). This may be attributed to inherently lower amounts of NO in the parasite or altered esterase activity or even variations in their membrane permeability.…”

Section: Resultsmentioning

confidence: 92%

“…Standardization of DAF-2DA Labeling in Leishmania Promastigotes DAF-2DA has previously been used for measurement of NO in a variety of mammalian cells including monocytes, neutrophils (28,29) human spermatozoa (30), and endothelial cells (31). DAF-2DA has also effectively been used in Zebrafish Danio rerio (32) and the parasite Schistosoma mansoni (33), but the status of NO within Leishmania parasites has not been established.…”

Section: Resultsmentioning

confidence: 99%

“…Evidence suggests that DAF-based probes can also react with oxidized derivatives of NO, e.g., peroxynitrite [ONOO 2 (29,38)], reductants [dehydroascorbic acid, ascorbic acid, dithiothreitol, 2-mercaptoethanol, and glutathione (39,40)] divalent cations [mainly calcium, (41)], and endogenous nitroso/nitrosyl species [i.e., nitrosothiols, nitrosamines, and heme-NO products, (29)], which could account for the residual DAF-2T fluorescence present after treatment with C-PTIO.…”

Section: Resultsmentioning

confidence: 99%

“…Mammalian cells require only 2.0 lM DAF-2DA for measurement of intracellular NO (28,29), whereas Leishmania parasites need a 20 fold higher concentration of DAF-2DA. Importantly, measurable amounts of fluorescence were detectable only after adding [10 lM DAF-2DA (Fig.…”

Section: Parasitized Macrophages Had Decreased Levels Of No That Was mentioning

confidence: 99%

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Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

et al. 2010

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Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 lM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 lM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds. ' International Society for Advancement of Cytometry Key termsanti-leishmanial; DAF-2DA; flow cytometry; leishmaniasis; nitric oxide LEISHMANIA are intracellular protozoan parasites that infect host mononuclear phagocytes, the resultant complex of diseases being leishmaniasis affecting 12 million people world wide in 88 countries (1). This disease is characterized by development of dermal lesions that may be cutaneous leishmaniasis (CL), diffuse cutaneous leishmaniasis (DCL), or mucocutaneous leishmaniasis (MCL) as also there can be systemic involvement as in the case of visceral leishmaniasis (VL), which is potentially fatal if left untreated (2).This obligate intracellular parasite resides and multiplies within macrophages, and therefore it should be able to evade the cytotoxic mechanisms innately operative within the macrophages for its survival (3). One of the predominant cytotoxic mechanisms includes production of reactive nitrogen intermediates, which the Leishmania parasite effectively decreases to ensure its survival (4,5).

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Parasitized Macrophages Had Decreased Levels Of No That Was mentioning

confidence: 99%

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Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

et al. 2010

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“…The compound DAF-2 DA is a membrane-permeable reagent, has a low detection limit (2 to 5 nM), and provides the opportunity for direct detection of intracellular NO (35). DAF-2 DA reaction with NO yields a green fluorescent signal that can be detected both qualitatively and quantitatively (16,36). We wanted to determine NO production during infection and in response to a classical macrophage activation signal important in the host response to intracellular pathogens.…”

Section: Resultsmentioning

confidence: 99%

The Intracellular Environment of Human Macrophages That Produce Nitric Oxide Promotes Growth of Mycobacteria

et al. 2013

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e Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-␥), L-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-␥ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.

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Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals

et al. 2010

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In the present work, a fluorimetric automatic method based on multisyringe flow injection analysis (MSFIA) was developed for in vitro evaluation of scavenging capacity against nitric oxide (NO) using 4,5-diaminofluorescein (DAF-2) as an NO-selective fluorogenic probe. The MSFIA manifold was assembled to perform the in-line generation of NO and the competitive reaction of putative scavenger molecules and DAF-2 with NO at conditions close to those found in vivo regarding temperature (37 degrees C), pH (7.4), and concentration of NO (less than 1 microM). This approach allowed the evaluation of scavenging capacity against NO by endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC(50) values were calculated for rutin (1.30 +/- 0.02 microM, positive control), cysteine (321 +/- 8 microM), reduced glutathione (1106 +/- 93 microM), uric acid (134 +/- 12 microM), dipyrone (1.36 +/- 0.06 microM), and captopril (363 +/- 28 microM). A high degree of automation was attained as the successive dilution of antioxidant standard solutions required for IC(50) assessment was performed automatically, in a dilution chamber placed in the flow system. A determination throughput of 16 h(-1) and a good precision were attained (relative standard deviation between 1.6 and 9.0%), fostering the application of the proposed method to routine/screening analysis of scavenging capacity against NO.

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

Cited by 19 publications

References 47 publications

Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

The Intracellular Environment of Human Macrophages That Produce Nitric Oxide Promotes Growth of Mycobacteria

Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals

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