Protocol for quantifying mitotic progression in adherent and suspension cells by live-cell imaging

Live-cell imaging is used to monitor mitotic progression in adherent cells; however, its application to suspension cells remains limited. Here, we present a protocol for quantifying mitotic progression in adherent and suspension cells by monitoring chromatin condensation using the live-cell DNA probe SPY-DNA. We describe steps for cell synchronization, cell plating, SPY-DNA labeling, and live-cell imaging. We then detail procedures for surface plot visualization and intensity profile analysis. This approach enables direct comparison of mitotic progression between adherent and suspension cells. For complete details on the use and execution of this protocol, please refer to Kamranvar et al.¹.