Development of cell-based immunoassays to measure type I collagen in cultured fibroblasts

Excessive deposition of type I collagen by activated fibroblasts is a hallmark of scarring and fibrotic pathologies. Quantitation of collagen I at the protein level is paramount to measure functionally relevant changes during pathological remodeling of the extracellular matrix. We describe two new c...

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Vydané v:The international journal of biochemistry & cell biology Ročník 42; číslo 11; s. 1808 - 1815
Hlavní autori: Jones, Brian, Bucks, Christine, Wilkinson, Patrick, Pratta, Michael, Farrell, Francis, Sivakumar, Pitchumani
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: Netherlands Elsevier Ltd 01.11.2010
Predmet:
Assaying
Beta
Blotting, Western
Cells, Cultured
Collagen
Collagen Type I - metabolism
Collagens
Density
Enzyme-Linked Immunosorbent Assay
Enzymes
Extracellular matrix
Extracellular Matrix - drug effects
Extracellular Matrix - metabolism
Fibroblasts
Fibroblasts - drug effects
Fibroblasts - metabolism
Fibrosis
Format
Human
Humans
Immunoassay - methods
Microscopy, Fluorescence
Radioimmunoassay
TGFβ
Transforming Growth Factor beta - pharmacology
NHLF
Collagen
Fibrosis
NHDF
TGFβ
Fibroblasts
Extracellular matrix
ICW
COL1
ECM
ELISA
ISSN:1357-2725, 1878-5875, 1878-5875
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Popis
Shrnutí:Excessive deposition of type I collagen by activated fibroblasts is a hallmark of scarring and fibrotic pathologies. Quantitation of collagen I at the protein level is paramount to measure functionally relevant changes during pathological remodeling of the extracellular matrix. We describe two new cell-based assays to directly quantify the amount of collagen I incorporated into the extracellular matrix of primary human lung fibroblasts. Utilizing a monoclonal antibody specific to native human collagen I, we optimized conditions and parameters including incubation time, specificity and cell density to demonstrate dose-dependent induction of collagen I by transforming growth factor beta, as measured by in-cell enzyme linked immunosorbent assay. The results obtained by this assay were mimicked by an “In situ Quantitative Western Blot” on cultured cells using the same antibody. Results from these assays were comparable to those obtained with a commercial assay for collagen I N-propeptide, which is an index of collagen formation. These assays have been optimized for a 96-well format and provide a novel and useful approach for screening of anti-fibrotic agents in vitro. The assays described here also offer a significant improvement in throughput and specificity over conventional methods that primarily measure soluble collagen.
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ISSN:1357-2725
1878-5875
1878-5875
DOI:10.1016/j.biocel.2010.07.011