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Profiling inflammatory responses with microfluidic immunoblotting.

ABSTRACT: Rapid profiling of signaling pathways has been a long sought after goal in biological sciences and clinical medicine. To understand these signaling pathways, their protein components must be profiled. The protein components of signaling pathways are typically profiled with protein immunoblotting. Protein immunoblotting is a powerful technique but has several limitations including the large sample requirements, high amounts of antibody, and limitations in assay throughput. To overcome some of these limitations, we have designed a microfluidic protein immunoblotting device to profile multiple signaling pathways simultaneously. We show the utility of this approach by profiling inflammatory signaling pathways (NF?B, JAK-STAT, and MAPK) in cell models and human samples. The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting. Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies. We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.

SUBMITTER: Chang HN

PROVIDER: S-EPMC3842271 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Profiling inflammatory responses with microfluidic immunoblotting.

Chang Huai-Ning HN Leroueil Pascale R PR Selwa Katherine K Gasper C J CJ Tsuchida Ryan E RE Wang Jason J JJ McHugh Walker M WM Cornell Timothy T TT Baker James R JR Goonewardena Sascha N SN

PloS one 20131127 11

Rapid profiling of signaling pathways has been a long sought after goal in biological sciences and clinical medicine. To understand these signaling pathways, their protein components must be profiled. The protein components of signaling pathways are typically profiled with protein immunoblotting. Protein immunoblotting is a powerful technique but has several limitations including the large sample requirements, high amounts of antibody, and limitations in assay throughput. To overcome some of the ...[more]

PMID: 24312374

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Project description:Inflammatory bladder disorders such as interstitial cystitis (IC) deserve attention since a major problem of the disease is diagnosis. IC affects millions of women and is characterized by severe pain, increased frequency of micturition, and chronic inflammation. Characterizing the molecular fingerprint (gene profile) of IC will help elucidate the mechanisms involved and suggest further approaches for therapeutic intervention. Therefore, in the present study we used established animal models of cystitis to determine the time course of bladder inflammatory responses to antigen, Escherichia coli lipopolysaccharide (LPS), and substance P (SP) by morphological analysis and cDNA microarrays. The specific aim of the present study was to compare bladder inflammatory responses to antigen, LPS, and SP by morphological analysis and cDNA microarray profiling to determine whether bladder responses to inflammation elicit a specific universal gene expression response regardless of the stimulating agent. During acute bladder inflammation, there was a predominant infiltrate of polymorphonuclear neutrophils into the bladder. Time-course studies identified early, intermediate, and late genes that were commonly up-regulated by all three stimuli. These genes included: phosphodiesterase 1C, cAMP-dependent protein kinase, iNOS, beta-NGF, proenkephalin B and orphanin, corticotrophin-releasing factor (CRF) R, estrogen R, PAI2, and protease inhibitor 17, NFkB p105, c-fos, fos-B, basic transcription factors, and cytoskeleton and motility proteins. Another cluster indicated genes that were commonly down-regulated by all three stimuli and included HSF2, NF-kappa B p65, ICE, IGF-II and FGF-7, MMP2, MMP14, and presenilin 2. Furthermore, we determined gene profiles that identify the transition between acute and chronic inflammation. During chronic inflammation, the urinary bladder presented a predominance of monocyte/macrophage infiltrate and a concomitant increase in the expression of the following genes: 5-HT 1c, 5-HTR7, beta 2 adrenergic receptor, c-Fgr, collagen 10 alpha 1, mast cell factor, melanocyte-specific gene 2, neural cell adhesion molecule 2, potassium inwardly-rectifying channel, prostaglandin F receptor, and RXR-beta cis-11-retinoic acid receptor. We conclude that microarray analysis of genes expressed in the bladder during experimental inflammation may be predictive of outcome. Further characterization of the inflammation-induced gene expression profiles obtained here may identify novel biomarkers and shed light into the etiology of cystitis.

Dataset Information

Profiling inflammatory responses with microfluidic immunoblotting.

Publications

Profiling inflammatory responses with microfluidic immunoblotting.

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