Reproducible Sample Preparation of Virus-Infected Cells for Cryo-FIB/ET Using Manual Plunge Freezing.

Abstract

Most viruses extensively remodel their host cells to establish productive infection. Visualization of virus-induced cellular remodeling by electron microscopy (EM) has been revolutionized in recent years by advances in cryo-focused ion beam (cryo-FIB) milling paired with cryo-electron tomography (cryo-ET). As cryo-FIB/ET becomes more widely available, there is a need for beginner-friendly guides to optimize the preparation of virus-infected mammalian cells on EM grids. Here, we provide an in-house protocol for new users for preparing samples of cells infected with herpes simplex virus 1 (HSV-1) for cryo-FIB/ET. This protocol guides users in how to seed infected cells onto grids, blot, and plunge-freeze grids using basic, manual equipment. It also provides tips on how to screen and prioritize grids for efficient milling and data collection. Key features • A beginner-friendly protocol for users without access to a cryo-EM core/suite at their institution that utilizes basic equipment. • This protocol focuses on optimizing cell seeding and blotting to yield grids with thin ice and evenly distributed cells. • Grids prepared using this protocol can be used for focused ion beam milling.