Protocol to quantify glioblastoma cell invasion and nuclear deformations in 3D hydrogels

Here, we present a protocol to quantify 3D invasion and nuclear deformation from time-lapse imaging of U251 glioblastoma spheroids embedded in Matrigel. We describe steps for computing a time-resolved radial invasion index through epifluorescence imaging and extracting per-nucleus deformation from 3D+time data using spinning-disk confocal microscopy with an optical flow-based pipeline. This adaptable protocol links nuclear mechanics to invasion. For complete details on the use and execution of this protocol, please refer to van Bodegraven et al.¹.