ProMAT: protein microarray analysis tool

White, A. M.; Daly, Don S.; Varnum, Susan M.; Anderson, Kevin K.; Bollinger, Nikki; Zangar, Richard C.

doi:10.1093/bioinformatics/btl093

Cited by 37 publications

(36 citation statements)

References 2 publications

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“…Recombinant protein standard curves were derived by measuring the intensity values of recombinant proteins at concentrations between 10 pg/ml and 100,000 pg/ml in the SCBC. The 4 Parameter Logistic nonlinear regression model was used to fit the standard curves, and the 95% confidence intervals were calculated with ProMat (47). Concentration values larger than the maximum recombinant protein concentration (100,000 pg/ml) were set to 100,000 pg/ml.…”

Section: Methodsmentioning

confidence: 99%

Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation

et al. 2015

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Cellular responses are mediated by heterogeneous intermediate signals that are secreted and sensed by the same cells. Cell-to-cell communication through these intermediate signals likely affects the collective response of cells within a population. We combined multiplexed, microwell-based measurements of cytokine secretion by single cells with data from the analysis of cell populations to determine the role of paracrine signaling in shaping the profile of inflammatory cytokines secreted by macrophages in response to the stimulation of Toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS). Loss of paracrine signaling as a result of cell isolation substantially reduced the secretion of a subset of LPS-stimulated cytokines, including interleukin-6 (IL-6) and IL-10, by macrophage-like U937 cells and human monocyte-derived macrophages (MDMs). Graphical Gaussian modeling (GGM) of the single-cell data defined a regulatory network of paracrine signals, which was validated experimentally in the population through antibody-mediated neutralization of individual cytokines. Tumor necrosis factor-α (TNF-α) was identified as the most influential cytokine in the GGM network, and our data suggest that paracrine signaling from a small subpopulation of “high-secreting” cells, which generated most of the TNF-α produced, was necessary but not sufficient to achieve high secretion of IL-6 and IL-10 in the cell population. Decreased IL-10 secretion in isolated MDMs was linked to increased TNF-α secretion, suggesting that inhibition of the inflammatory response also depends on paracrine signaling. Our results reveal a previously uncharacterized role for cell-to-cell communication within a population in coordinating a rapid and reliable innate immune response in spite of underlying cell-to-cell heterogeneity.

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Section: Methodsmentioning

confidence: 99%

Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation

et al. 2015

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“…Following exposure of equal numbers of cells to silica ENPs in serum-free medium at the concentrations indicated, the medium was collected, centrifuged to remove particles and cell debris, and diluted up to 10-fold for analysis. Standard curves were generated and sample antigen concentrations were calculated using the Protein Microarray Analysis Tool software (White et al 2006). Five separate culture dishes were analyzed at each ENP concentration and results are reported as the mean protein concentration ± standard error.…”

Section: Protein Microarray Elisa Analysismentioning

confidence: 99%

Cellular recognition and trafficking of amorphous silica nanoparticles by macrophage scavenger receptor A

Orr¹,

Chrisler²,

Cassens³

et al. 2010

Nanotoxicology

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The cellular uptake of engineered nanoparticles (ENPs) is known to involve active transport mechanisms, yet the biological molecules involved are poorly understood. We demonstrate that the uptake of amorphous silica ENPs by macrophage cells, and the secretion of proinflammatory cytokines, is strongly inhibited by silencing expression of scavenger receptor A (SR-A). Conversely, ENP uptake is augmented by introducing SR-A expression into human cells that are normally non-phagocytic. Confocal microscopy analyses show that the majority of single or small clusters of silica ENPs co-localize with SR-A and are internalized through a pathway characteristic of clathrin-dependent endocytosis. In contrast, larger silica ENP agglomerates (>500 nm) are poorly co-localized with the receptor, suggesting that the physical agglomeration state of an ENP influences its cellular trafficking. As SR-A is expressed in macrophages throughout the reticulo-endothelial system, this pathway is likely an important determinant of the biological response to ENPs.

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“…Both ProMAT Calibrator and ProMAT are compatible with ELISA-BASE . ELISA-BASE, ProMAT Calibrator and ProMAT are freely distributed with a GNU-like license as open-source, open-development software via web-distributed, self-installing packages (White et al, 2007). We have previously described ProMAT Calibrator, including the development and validation of a calibrant assay for green fluorescent protein, in an article targeted towards biologists and other expected end users ).…”

Section: Predicting Protein Concentrationsmentioning

confidence: 99%

An Internal Calibration Method for Protein-Array Studies

Daly¹,

Anderson²,

Seurynck-Servoss³

et al. 2010

Statistical Applications in Genetics and Molecular Biology

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Nuisance factors in a protein-array study add obfuscating variation to spot intensity measurements, diminishing the accuracy and precision of protein concentration predictions. The effects of nuisance factors may be reduced by design of experiments, and by estimating and then subtracting nuisance effects. Estimated nuisance effects also inform about the quality of the study and suggest refinements for future studies.We demonstrate a method to reduce nuisance effects by incorporating a non-interfering internal calibration in the study design and its complemental analysis of variance. We illustrate this method by applying a chip-level internal calibration in a biomarker discovery study. The variability of sample intensity estimates was reduced 16% to 92% with a median of 58%; confidence interval widths were reduced 8% to 70% with a median of 35%. Calibration diagnostics revealed processing nuisance trends potentially related to spot print order and chip location on a slide. The accuracy and precision of a protein-array study may be increased by incorporating a non-interfering internal calibration. Internal calibration modeling diagnostics improve confidence in study results and suggest process steps that may need refinement. Though developed for our protein-array studies, this internal calibration method is applicable to other targeted array-based studies.

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ProMAT: protein microarray analysis tool

Abstract: ProMAT is available at http://www.pnl.gov/statistics/ProMAT. ProMAT requires Java version 1.5.0 and R version 1.9.1 (or more recent versions). ProMAT requires either Windows XP or Mac OS 10.4 or newer versions.

Cited by 37 publications

References 2 publications

Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation

Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation

Cellular recognition and trafficking of amorphous silica nanoparticles by macrophage scavenger receptor A

An Internal Calibration Method for Protein-Array Studies

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