Protocol for quantitative image analysis of alizarin red staining to assess osteogenesis in cultured cells

Endothelial-to-mesenchymal transition (EndMT) generates mesenchymal-like cells with multilineage differentiation potential. We describe steps for inducing EndMT in human venous endothelial cells, directing their differentiation toward an osteogenic lineage, and assessing mineralization using an alizarin red assay. We then detail procedures for quantifying and classifying osteogenic capacity from alizarin red staining using custom image analysis pipelines. This protocol can also be used to quantify alizarin red staining in mouse-derived mesenchymal stem/stromal cells (MSCs) in vitro. For complete details on the use and execution of this protocol, please refer to Kishta et al.¹.