Maassen Sjors, Warner Harry M, Grijpstra Pieter, van den Bogaart Geert
Department of Molecular Immunology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Groningen, the Netherlands.
Department of Medical Biology and Pathology, University Medical Centre Groningen, Groningen, the Netherlands.
MethodsX. 2023 Jul 17;11:102288. doi: 10.1016/j.mex.2023.102288. eCollection 2023 Dec.
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.
胶原蛋白重塑是胚胎发育和成年机体稳态维持的重要过程。胶原蛋白重塑是成纤维细胞、巨噬细胞和其他细胞中的一个复杂过程，在此过程中，新的胶原蛋白被分泌并聚合成纤维，而旧的胶原蛋白则通过蛋白水解和胞吞作用被清除。虽然胶原蛋白的产生已得到充分研究，但胶原蛋白的清除却了解较少。在本方案中，我们描述了一种细胞摄取胶原蛋白定量方法。该方案基于I型胶原蛋白 - 异硫氰酸荧光素（FITC）偶联物在细胞培养板孔中的聚合。接下来，洗去未聚合的胶原蛋白，并在细胞培养基中加入细胞。在此阶段，如果需要，可对其进行抑制剂和/或刺激剂处理。之后，使用蛋白酶Accutase将细胞从胶原蛋白上分离，并使用显微镜和/或流式细胞术对FITC信号进行定量。
•用于定量测量细胞摄取胶原蛋白的易于使用的方案。
•使用Accutase可快速轻松地使细胞与胶原蛋白分离，即使对于像巨噬细胞这样强粘附的细胞也是如此。
•下游应用可以是多种分析技术，如显微镜检查、RNA和蛋白质分离以及流式细胞术。
