Computational protocol: CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules in the following Mitosis

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[…] Cells subjected to nocodazole shock were permeabilized for 20 s at room temperature in 1x PermFix (100 mM K-PIPES, pH 6.8, 0.4% Triton-X 100, 10mM K-EGTA, 1mM MgCl2) immediately before fixation. All cells were fixed in methanol at -20°C for 10 min. Rinses were performed with TBS+0.1% Triton-X 100 (TBST). Coverslips were blocked with AbDil (TBST + 2 mg/mL bovine serum albumin (Sigma-Aldrich)) for 10 min, probed with primary antibodies diluted in AbDil for 1 h, rinsed, probed with secondary antibodies diluted in AbDil for 45 min, and rinsed. DNA was stained with 5 μg/mL Hoechst 33342. Coverslips were mounted in Prolong Gold (Invitrogen).For all figures except , fixed cells were imaged using a 60X 1.4 NA objective (Olympus) with 1.6X auxiliary magnification on a DeltaVision Elite imaging system (Applied Precision). Optical sections were collected every 0.2 μm. Ratio deconvolution and maximum-Z projection were performed in SoftWorx (Applied Precision). Images were prepared for publication using ImageJ and NIS-Elements (Nikon) software. All images shown are maximum-Z projections of deconvolved image stacks. For a given panel, all images are shown with the same look-up table (LUT) applied.For , fixed cells were imaged using a 20X 0.45 NA objective (Olympus) on a DeltaVision Elite imaging system (Applied Precision). Optical sections were collected every 1 μm. Maximum-Z projection was performed in SoftWorx (Applied Precision). Images were prepared for publication using ImageJ and NIS-Elements (Nikon) software. All images shown are maximum-Z projections of image stacks. For a given panel, all images are shown with the same look-up table (LUT) applied.Quantification of spindle intensity was performed in ImageJ: for a single, central Z-plane, the average fluorophore intensity of the non-spindle cytoplasm was subtracted from the average fluorophore intensity of the spindle to give the background-subtracted spindle intensity; the background-subtracted spindle intensity of the relevant spindle-binding protein (Kif15 or HURP) was then expressed as a ratio over the background-subtracted spindle intensity of tubulin. Quantification of residual K-MT polymer following nocodazole shock was performed in ImageJ: most of the background was subtracted using the rolling ball algorithm (radius 10 pixels); for each slice, the average tubulin intensity of the K-MT region minus the average residual background intensity measured from an acellular region was measured and multiplied by the area of the K-MT region to give the total tubulin intensity for the slice; and this total tubulin intensity was summed over the Z-stack. Because some cells had significant unpolymerized tubulin staining at the cell edge, the K-MT region for intensity measurements was drawn to avoid this nonspecific signal. [...] For figure panels 1C, 1D, 1E, 1J, 2E, 4D, 5B, 5C, and 5F, T-tests (two-tailed, assuming unequal variance) were performed with the TTEST function in Excel (Microsoft). For figure panels 2F, 2H, and (panel B), a one-way ANOVA with Tukey test was performed in SigmaPlot (Systat Software). For figure panels 2C, 2D, 3A, 3C, 4B, and 4C, a two-way ANOVA with Tukey test was performed in SigmaPlot. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens