Quantitative imaging of single-cell phenotypes in cancer cells cultured on hydrogel surfaces

Small interfering RNA (siRNA) screening approaches used with quantitative single-cell analysis can uncover the roles of genes in cell morphogenesis. Here, we present a high-throughput automated phenotypic screening technique to quantify a single cell shape in cancer cells cultured on top of soft 3D hydrogels. We describe reverse transfection of cells with siRNAs and seeding of these cells on top of collagen, followed by image analysis to quantify morphology of a single cell and population levels in low-elasticity matrices. For complete details on the use and execution of this protocol, please refer to Bousgouni et al. (2022).¹.