An Integrated Microfluidic Device for Monitoring Changes in Nitric Oxide Production in Single T-Lymphocyte (Jurkat) Cells

Metto, Eve C.; Evans, Karsten; Barney, Patrick; Culbertson, Anne H.; Gunasekara, Dulan B.; Caruso, Giuseppe; Hulvey, Matthew K.; Silva, José Alberto Fracassi da; Lunte, Susan M.; Culbertson, Christopher T.

doi:10.1021/ac401665u

Cited by 42 publications

(43 citation statements)

References 58 publications

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“…In most instances, the beneficial effects of Car treatment were related to its generally ascribed antioxidant activity [31], which involves a capacity to decrease both ROS and RNS in different experimental models [32, 33]. In agreement with these and other studies, our experiments with cultured murine RAW 264.7 macrophages, using ME-LIF to detect the stable fluorescent NO adduct in cell lysates [27, 34], confirmed that Car decreases NO concentration in stimulated cells in a dose-dependent manner (Fig. 1 and Online Resource 1).…”

Section: Discussionsupporting

confidence: 70%

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages

et al. 2017

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Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation, and is generally accompanied by increased oxidative/nitrosative stress. Carnosine (β-alanine-histidine (β-Ala-His)) has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity end product, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (β-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages, the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of carnosine that modulate the NO/nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.

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Section: Discussionsupporting

confidence: 70%

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages

et al. 2017

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“…We have already reported a single cell chemical cytommetric device for NO detection from Jurkat cells using a NO-selective fluorophore. 29 The ultimate goal is to use ME-EC to measure multiple redox-active species in a single cell as an indication of nitrosative stress.…”

Section: Resultsmentioning

confidence: 99%

Microchip electrophoresis with amperometric detection method for profiling cellular nitrosative stress markers

Gunasekara

Siegel

Caruso

et al. 2014

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Summary The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2−) in cell lysates. There was a 2.5- to 4-fold increase in NO2− production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2− inside a single unstimulated macrophage cell was estimatedto be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions.

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“…PDMS electrophoresis microchips were fabricated by soft lithography using a high‐relief master previously defined in silicon wafer as described elsewhere . Briefly, PDMS monomer and the curing agent were mixed at a ratio of 10:1 m/m, poured then on the master, and kept at 70ºC during 30 min.…”

Section: Methodsmentioning

confidence: 99%

Metalless electrodes for capacitively coupled contactless conductivity detection on electrophoresis microchips

et al. 2015

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This paper describes the use of ionic solutions as sensing electrodes for capacitively coupled contactless conductivity detection on electrophoresis microchips. Initially, two channels were engraved in a PMMA holder by using a CO2 laser system and sealed with a thin adhesive membrane. PDMS electrophoresis chips were fabricated by soft lithography and reversibly sealed against the polymer membrane. Different ionic solutions were investigated as metalless electrodes. The electrode channels were filled with KCl solutions prepared in conductivity values from approximately 10 to 40 S/m. The best analytical response was achieved using the KCl solution with 21.9 S/m conductivity (2 mol/L). Besides KCl, we also tested NaCl and LiCl solutions for actuating as detection electrodes. Taking into account the same electrolyte concentration (2 mol/L), the best response was recorded with KCl solution due to its higher ionic conductivity. The optimum operating frequency (400 kHz) and the best sensing electrode (2 mol/L KCl) were used to monitor electrophoretic separations of a mixture containing K(+) , Na(+) , and Li(+) . The use of liquid solutions as sensing electrodes for capacitively coupled contactless conductivity detection measurements has revealed great performance to monitor separations on chip-based devices, avoiding complicated fabrication schemes to include metal deposition and encapsulation of electrodes. The LOD values were estimated to be 28, 40, and 58 μmol/L for K(+) , Na(+) , and Li(+) , respectively, what is comparable to that of conventional metal electrodes. When compared to the use metal electrodes, the proposed approach offers advantages regarding the easiness of fabrication, simplicity, and lower cost per device.

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An Integrated Microfluidic Device for Monitoring Changes in Nitric Oxide Production in Single T-Lymphocyte (Jurkat) Cells

Cited by 42 publications

References 58 publications

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages

Microchip electrophoresis with amperometric detection method for profiling cellular nitrosative stress markers

Metalless electrodes for capacitively coupled contactless conductivity detection on electrophoresis microchips

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