Preparation of Vitrified Mammalian Cells for <i>In Situ</i> Cryo‐Electron Tomography

In situ cryo-electron tomography (cryo-ET) is a rapidly developing approach that enables three-dimensional imaging of cryogenically preserved mammalian cells at up to subnanometer resolution under near-native conditions. Integral to these methods is the successful culture and vitrification of cells directly on electron microscopy (EM) grids. Previous approaches have been individualized for specific cell types. Here, we describe a generalizable protocol applicable to both immortalized and primary mammalian cells. We specifically focus on cryo-ET sample preparation using INS-1E cells, an immortalized rat pancreatic beta-cell line, and primary human fibroblasts. The protocol ensures efficient application and culture of adherent mammalian cells onto EM grids. We then describe the vitrification process by which cultured cells on grids are cryo-preserved in vitreous ice for subsequent cryo-ET imaging. This protocol offers a streamlined approach to mammalian cell-based sample preparation for in situ cryo-ET. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Culture and vitrification of mammalian cells on electron microscopy grids.