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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a 2-fold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.85211018810195NIH [R21NS061202]Terry Johnson Cancer Center, Kansas State UniversityDOD ASSURE [CHE-1004991]University of Catania, ItalyAmerican Heart AssociationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)NIH [R21NS061202]DOD ASSURE [CHE-1004991]FAPESP [2010/01046-6

Monitoring intracellular nitric oxide production using microchip electrophoresis and laser-induced fluorescence detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Nitric oxide (NO) is a biologically important short-lived reactive species that has been shown to be involved in a large number of physiological processes. The production of NO is substantially increased in immune and other cell types through the upregulation of inducible nitric oxide synthase (iNOS) caused by exposure to stimulating agents such as lipopolysaccharide (LPS). NO production in cells is most frequently measured via fluorescence microscopy using diaminofluorescein-based probes. Capillary electrophoresis with laser-induced fluorescence detection has been used previously to separate and quantitate the fluorescence derivatives of NO from potential interferences in single neurons. In this paper, microchip electrophoresis (ME) coupled to laser-induced fluorescence (LIF) detection is evaluated as a method for measurement of the NO production by Jurkat cells under control and stimulating conditions. ME is ideal for such analyses due to its fast and efficient separations, low volume requirements, and ultimate compatibility with single cell chemical cytometry systems. In these studies, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) was employed for the detection of NO, and 6-carboxyfluorescein diacetate (6-CFDA) was employed as an internal standard. Jurkat cells were stimulated using lipopolysaccharide (LPS) to produce NO, and bulk cell analysis was accomplished using ME-LIF. Stimulated cells exhibited an approximately 2.5-fold increase in intracellular NO production compared to the native cells. A NO standard prepared using diethylamine NONOate (DEA/NO) salt was used to construct a calibration curve for quantitation of NO in cell lysate. Using this calibration curve, the average intracellular NO concentrations for LPS-stimulated and native Jurkat cells were calculated to be 1.5 mM and 0.6 mM, respectively.42414420NIH [R01 NS042929, R21 NS061202]NIH NCRR [P20RR016475]Adams InstituteAmerican Heart AssociationUniversity of Catania, ItalyFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)NIH [R01 NS042929, R21 NS061202]NIH NCRR [P20RR016475]FAPESP [2010/010466

Who Cares What Who Prefers? A Study in Judgment Differences Between Syntacticians and Non-syntacticians

This exploratory study contributes to the discussion of possible differences in the syntactic judgments of experts and non-experts. In particular, we investigated whether experts in a narrow sense (syntacticians) and experts in a broader sense (linguists not specializing in syntax) react differently to superiority violations in embedded clauses (who wonders what who saw) in an interpretation preference task. The overall result supports syntactic models that deal with superiority violations in terms of a competition between alternative expressions of the same meaning. The effects show up clearly in the judgment patterns of experts in the narrow sense (syntacticians) only, and thus point to the existence of a judgment difference among linguists from different subfields. This can lend support to an explanation in terms of shallow versus deep processing of complex syntactic structures