A protocol for fast observation and study of the nuclear surface ultrastructure in adherent cultured somatic cells

The nucleus is the largest organelle in eukaryotic cells and is involved in genome storage, signal transduction, material transport and virus replication. Dissecting the ultrastructure of the nuclear surface facilitates a more comprehensive understanding of the nuclear structure and function, particularly the nuclear pore complex (NPC). In this protocol, we describe detailed procedures for rapidly observing the nuclear surface ultrastructure of adherent somatic cells, including cell culture and treatment, nuclear isolation via centrifugation, and sample preparation for scanning electron microscopy (SEM). Cells are treated with nocodazole and cytochalasin B (CB) to depolymerize microtubules and actin ϐilaments, thereby loosening nucleo-cytoskeletal connections, followed by low-speed centrifugation to obtain nuclei without ultracentrifugation. We further compared NPC structures from different cells and calculated the number of NPCs in three distinct types of cells. Compared to conventional nuclear isolation methods, our protocol is faster and gentler. Quantitative morphometric analyses demonstrate that our protocol signiϐicantly improves nuclear circularity and surface smoothness, as well as NPC circularity and aspect ratio (AR), while preserving mean NPC diameter. Moreover, our method reduces variability in NPC pore area, indicating more uniform preservation. Collectively, these quantitative data show that our protocol achieves signiϐicantly improved preservation of nuclear morphology and NPC ultrastructure compared to conventional methods.