Resume

• Chemistry

  FTIR

  Spectroscopy

  HPLC

  NMR

  Cell Culture

  Mass Spectrometry

  UV/Vis

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

  Culbertson AH

  Barney P

  Evans K

  José Alberto Fracassi

  Sue Lunte

  Dulan B. Gunasekara

  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 twofold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.

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

  Sue Lunte

  Christopher T. Culbertson

  Anne H. Culbertson

  Jose Alberto Fracassi da Silva

  Matthew K. Hulvey

  Derek T. Jensen

  Dulan B. Gunasekara

  Emilie R. Mainz

  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.

  Ph-D Student

  Culbertson AH

  Barney P

  Evans K

  José Alberto Fracassi

  Sue Lunte

  Dulan B. Gunasekara

  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 twofold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.

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

  University of Minnesota Duluth

  Kansas State University

  kansas State University

  Manhattan

  KS

  Graduate Research Assistant

  Kansas State University

  University of Minnesota Duluth

  Egerton University

  Bachelor's Degree

  Secondary Education and Teaching

  Kansas State University

  PhD Analytical Chemistry