Optimization of Reporter Cells for Expression Profiling in a Microfluidic Device

Wieder, Kenneth J.; King, Kevin R.; Thompson, Deanna M.; Zia, Cindy; Yarmush, Martin L.; Jayaraman, Arul

doi:10.1007/s10544-005-3028-3

Cited by 47 publications

(40 citation statements)

References 28 publications

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“…Both GFP and luciferase are widely used reporter genes [30]. GFP has the advantage of requiring no processing as its expression can be monitored at single cell basis, which facilitates screening by FACS.…”

Section: Discussionmentioning

confidence: 99%

Potential problems inherent in cell-based stable NF-κB–GFP reporter systems

El-Guendy

Sinai

2008

Mol Cell Biochem

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The nuclear factor-κB (NF-κB) family of transcription factors plays a central role in numerous physiological processes including development, cell survival, immunity and inflammation. We generated a series of stable clonal lines in mouse embryonic fibroblasts carrying NF-κB-GFP plasmid as a reporter. These cell lines were selected by flow cytometry for their high responsiveness to tumor necrosis factor (TNFα) or lipopolysaccharide (LPS), two classic NF-κB inducing stimuli. Although all clones were generated from the same parental cell line, they each had a distinctive pattern of response to NF-κB stimuli. While exhibiting distinct profiles with regard to the GFP reporter, analysis of endogenous NF-κB downstream targets did not always show the same variability. This suggests that in the absence of confirmation of the signaling outcomes using endogenous outputs, considerable caution must be exercised in the interpretation of data using stable reporter systems.

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Section: Discussionmentioning

confidence: 99%

Potential problems inherent in cell-based stable NF-κB–GFP reporter systems

El-Guendy

Sinai

2008

Mol Cell Biochem

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“…Microfluidic devices were fabricated, as previously described (16,27,31), in the Materials Characterization Facility at Texas A&M University. Briefly, device designs were drawn in AutoCAD and used to create a high-resolution (Ͼ3,000 dots per inch) photolithography mask produced with a laser printer (Advanced Reproductions, North Andover, MA).…”

Section: ϩmentioning

confidence: 99%

Flow-Based Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable, Competing Gradients

Englert¹,

Manson²,

Jayaraman

2009

Appl Environ Microbiol

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108

114

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Chemotaxis is the migration of cells in gradients of chemoeffector molecules. Although multiple, competing gradients must often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to gradients of multiple signals. In this work, we developed a microfluidic device for generating precise and stable gradients of signaling molecules. We used the device to investigate the effects of individual and combined chemoeffector gradients on Escherichia coli chemotaxis. Laminar flow-based diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green fluorescent protein were determined using fluorescence microscopy. Quantification of the migration profiles indicated that E. coli was attracted to the quorum-sensing molecule autoinducer-2 (AI-2) but was repelled from the stationary-phase signal indole. Cells also migrated toward higher concentrations of isatin (indole-2,3-dione), an oxidized derivative of indole. Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between attractant and repellent signals may be important determinants of bacterial colonization of the gut.Bacteria sense chemoeffectors using cell surface receptors (13,29). Cells constantly monitor the concentration of specific molecules, comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement (6,22). Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Natural habitats for Escherichia coli, such as the gastrointestinal (GI) tract, are typically heterogeneous and contain multiple chemoeffectors with potentially opposing effects. The integrated chemotactic response in such environments is thus likely to be an important factor in bacterial colonization.Conventional approaches for investigating bacterial chemotaxis, such as the swim plate and capillary (1) assays, are not ideal for quantifying bacterial migration. Chemotactic-ring formation in semisolid agar requires metabolizable attractants and is subject to multiple factors, and both it and the traditional capillary assay are poorly designed to investigate repellent taxis. Mao et al. (23) were the first to investigate bacterial taxis in a microfluidic flow cell. In their device, a concentration gradient is formed by the diffusive mixing of two inlet streams. However, the exposure to a fully developed gradient in this device is limited because it takes time for the gradient to develop.Variations of this technique, such as three-channel microfluidic devices (7,8) in which a linear gradient is generated in the absence of flow or a T-channel device that monitors chemotaxis perpendicular to the direction of fluid flow (18), were developed subsequently. The T-channel system has many of the limitations of the device developed by Mao et al. (23), and nonflow systems,...

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“…For example, fluorescecent reporter genes can be introduced into cells in a model liver sinusoid such as those described above [130,131]. Addressed by various stimuli (drug, toxin, cytokine) using microfluidics, the cells would respond by emitting a fluorescent signal following reporter gene activation (i.e., NFκ-b).…”

Section: Current Challenges and Opportunitiesmentioning

confidence: 99%