Multi-tilt EM tomogram of a human bone osteosarcoma (U2OS) cell in the mitotic anaphase state. The tomogram refers to the U2OS cell in Figure 6 of the published manuscript: http://science.sciencemag.org/content/357/6349/eaag0025.
A human bone osteosarcoma cell in anaphase mitotic state was fixed with 2.5% glutaraldehyde, 5 mM CaCl2 in 0.1 M sodium cacodylate buffer (pH 7.4). Cells were stained with 10 µM DRAQ5, and photo-oxidized at 620 ± 30 nm for 3 minutes. Cells were post-fixed with 2% osmium tetroxide, 2 mM CaCl2, 1.5% potassium ferrocyanide in 0.15 M sodium cacodylate buffer (pH 7.4). Samples were ethanol dehydrated, embedded in Durcupan epoxy resin and sectioned to 250 nm thickness and mounted on copper slot grid. 5 nm and 10 nm colloidal gold particles were deposited on the grid as fiducial markers. 8-tilt EM tomography was collected on a 300 kV FEI Titan Krios using a 4K by 4K Gatan Ultrascan CCD camera at 1 degree increment for each tilt series from -60° to +60°. The entire set of EMT images was aligned and reconstructed using the Transform-based Tracking, Bundle adjustment, and Reconstruction (TXBR) package. The data has a voxel size of 1.6 nm.
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