A simple and effective method for ultrastructural analysis of mitosis in Drosophila S2 cells

Abstract

© 2016 The AuthorsThe Drosophila S2 tissue culture cells are a widely used system for studies on mitosis. S2 cells are particularly sensitive to gene silencing by RNA interference (RNAi), allowing targeted inactivation of mitotic genes. S2 cells are also well suited for high-resolution light microscopy analysis of mitosis in fixed cells, and can be easily immunostained to detect mitotic components. In addition, S2 cells are amenable to transformation with plasmid encoding fluorescently tagged mitotic proteins, allowing in vivo analysis of their behavior throughout cell division. However, S2 cells have not been widely used for transmission electron microscopy (TEM) analysis, which provides ultrastructural details on the morphology of the mitotic apparatus that cannot be obtained with high-resolution confocal microscopy. Here, we describe a simple method for the ultrastructural analysis of mitosis in Drosophila S2 cells. • Our method, which involves fixation and sectioning of a cell pellet, provides excellent preservation of mitotic structures and allows analysis of a higher number of mitotic divisions per sample, compared to correlative light-electron microscopy.• Dividing cells are randomly oriented within the pellet and are sectioned along different planes, providing all-around information on the structure of the mitotic apparatus.