Quantitative and organisational changes in mature extracellular matrix revealed through high-content imaging of total protein fluorescently stained in situ.
Sci Rep
; 7(1): 9963, 2017 08 30.
Article
em En
| MEDLINE
| ID: mdl-28855577
ABSTRACT
Fibrosis is a common driver of end-stage organ failure in most organs. It is characterised by excessive accumulation of extracellular matrix (ECM) proteins. Therapeutic options are limited and novel treatments are urgently required, however current cell-based high-throughput screening (HTS) models to identify molecules affecting ECM accumulation are limited in their relevance or throughput. We report a novel sensitive approach which combines in situ fluorescent staining of accumulated decellularised ECM proteins with automated high-content microscopy. Using this method to measure ECM accumulation in a kidney cell model, we demonstrated good agreement with established radiolabelled amino acid incorporation assays TGFß1 delivered a potent pro-fibrotic stimulus, which was reduced by TGFß antibody or the anti-fibrotic nintedanib. Importantly, our method also provides information about matrix organisation the extent of ECM accumulation was unaffected by the BMP antagonist Gremlin-1 but a pronounced effect on matrix fibrillar organisation was revealed. This rapid, straightforward endpoint provides quantitative data on ECM accumulation and offers a convenient cross-species readout that does not require antibodies. Our method facilitates discovery of novel pro- and anti-fibrotic agents in 384-well plate format and may be widely applied to in vitro cell-based models in which matrix protein deposition reflects the underlying biology or pathology.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fibrose
/
Proteínas
/
Matriz Extracelular
/
Nefropatias
/
Microscopia de Fluorescência
Limite:
Humans
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article