A quantitative in vitro collagen uptake assay

Abstract

Collagen remodelling is a vital process for embryonic development and homoeostatic maintenance of the adult body. Collagen remodelling is a complex process in fibroblasts, macrophages and other cells, whereby new collagen is secreted and polymerized into fibrils and old collagen is removed by proteolysis and endocytosis. Whereas the production of collagen is well-studied, the removal of collagen is less understood. In this protocol, we describe a method for the quantification of collagen uptake by cells. This protocol is based on the polymerisation of collagen type I-FITC conjugate in cell culture plate wells. Next, unpolymerized collagen is washed away and the cells are added in cell culture media. At this stage, they can be treated with inhibitors and/or stimulants if required. Afterwards, the cells are detached from the collagen using the protease accutase and the FITC signal is quantified using microscopy and/or flow cytometry. • Easy-to-use protocol for the quantitative measurement of collagen uptake in cells. • Cell detachment from collagen is quick and easy with accutase, even with strong adhering cells like macrophages. • Downstream applications can be a wide selection of analysis techniques like microscopy, RNA- and protein isolation, and flow cytometry.

Keywords

• A quantitative in vitro collagen uptake assay