Abstract
The 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.
Highlights
Light microscopy observation allows the choice of a region of interest in a block, which can be trimmed to obtain serial sections for transmission electron microscopy (TEM) analysis. This procedure significantly reduces the time required for collecting the data that are necessary for the ultrastructural analysis of the mitotic phase under study
Drosophila S2 cells serve as an excellent model system for a variety of cells biological analyses and high-throughput screens at genome-wide level [12]
S2 cells are a spontaneously immortalized cell line from a primary culture of Drosophila embryonic cells; their characteristics suggest that they are derived from macrophages [6]
Summary
All procedures should be performed at room temperature (23 Æ 2 C) unless otherwise specified. Post-fix the pellet for 1 h, continuously shaking (in vertically placed tube, on a regular laboratory horizontal shaker) at 20 rpm in 1% solution of osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4; see above), containing few crystals of potassium ferricyanide (K3[Fe(CN)6]) that makes the solution yellowish After this step, no further centrifugation is needed. Use the following step-by-step embedding protocol: Incubate the cell pellet in 1 ml of 1:3 (v/v) mixture of resin:acetone for 1 h, with continuous horizontal shaking (in a vertically placed tube on a regular laboratory horizontal shaker) at 20 rpm. Light microscopy observation allows the choice of a region of interest in a block, which can be trimmed to obtain serial sections for TEM analysis This procedure significantly reduces the time required for collecting the data that are necessary for the ultrastructural analysis of the mitotic phase under study
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