title: An integrated microfluidic device for monitoring changes in nitric oxide production in single T-lymphocyte (Jurkat) cells.
abstract: 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.
authors.affiliation: Department of Chemistry, Kansas State University , Manhattan, Kansas 66506, United States.
authors.forename: Eve C
authors.initials: EC
authors.lastname: Metto
authors.forename: Karsten
authors.initials: K
authors.lastname: Evans
authors.forename: Patrick
authors.initials: P
authors.lastname: Barney
authors.forename: Anne H
authors.initials: AH
authors.lastname: Culbertson
authors.forename: Dulan B
authors.initials: DB
authors.lastname: Gunasekara
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Caruso
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Hulvey
authors.forename: Jose Alberto
authors.initials: JA
authors.lastname: Fracassi da Silva
authors.forename: Susan M
authors.initials: SM
authors.lastname: Lunte
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Culbertson
mesh_terms: D006801:Humans; D019169:Jurkat Cells; D044085:Microfluidics / Q000295:instrumentation*; D009569:Nitric Oxide / Q000096:biosynthesis*; D012015:Reference Standards; D059010:Single-Cell Analysis; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D009569:Nitric Oxide