For live cell imaging, cells were plated in four-well or eight-well chambered glass-bottom slides (LabTekII) or 24-well glass-bottom plates (MatTek), transfected and imaged in a heated chamber (37°C and 5% CO2) using a ×10/0.3NA CPlanFLN or ×20/0.5NA UPLFLN objective on a Olympus IX-81 microscope, controlled by Cell-M software (Olympus). Images were acquired using a Hamamatsu ORCA-ER camera and processed using Cell-M software.
For immunofluorescence studies, cells plated on 12-mm coverslips, were pre-extracted with 0.2% Triton X-100 in PEM (100 mM PIPES (pH 6.8), 1 mM MgCl2 and 5 mM EGTA) for 45 s before fixation with 4% paraformaldehyde in PBS (for Mps1, Mad2 and Hec1). Aurora B and pAurB stainings were performed on cells fixed directly in 4% paraformaldehyde. Coverslips were blocked with 2% bovine serum albumin in PBS/0.1% Triton for 15 min, incubated with primary antibody for 16 h at 4 °C, washed with PBS, and incubated with secondary antibodies and 4,6-diamidino-2-phenylindole for an additional 2 h at room temperature. Coverslips were washed and mounted using ProLong antifade (Molecular Probes). All images were acquired on a DeltaVision RT system (Applied Precision) with a ×100/1.40NA UPlanSApo objective (Olympus) using SoftWorx software. Images are maximum intensity projections of deconvolved stacks.
For quantification of immunostaining, all images of similarly stained experiments were acquired with identical illumination settings and analysed using ImageJ. An ImageJ macro was designed to threshold and select all centromeres and all chromosomes areas (excluding centromeres), using the 4,6-diamidino-2-phenylindole and ACA channels. The convolve filter was first applied to the ACA channel and the threshold selection increased by 1 pixel (to ensure complete kinetochore selection). This was used to calculate the relative mean kinetochore intensity of Mad2, Mps1 and Hec1 ([centromere–chromosome arm intensity (Mad2/Mps1/Hec1)]/[centromere–chromosome arm intensity (ACA)]). For centromere localization of AurB/pAurB, a true background outside the cell was used to subtract from the centromere intensity (and centromere areas were enlarged by 3 pixels to incorporate the more diffuse Aurora B staining).
For quantification of cyclin B–mCherry fluorescence, ImageJ was used to calculate the total integrated fluorescence intensity of individual cells at each time point (after background subtraction of all images using a rolling ball radius of 200 pixels).
For all kinetochore intensity quantifications, means and standard deviations were calculated for each individual experiment and then combined using the standard method for non-overlapping data sets.
For immunofluorescence studies, cells plated on 12-mm coverslips, were pre-extracted with 0.2% Triton X-100 in PEM (100 mM PIPES (pH 6.8), 1 mM MgCl2 and 5 mM EGTA) for 45 s before fixation with 4% paraformaldehyde in PBS (for Mps1, Mad2 and Hec1). Aurora B and pAurB stainings were performed on cells fixed directly in 4% paraformaldehyde. Coverslips were blocked with 2% bovine serum albumin in PBS/0.1% Triton for 15 min, incubated with primary antibody for 16 h at 4 °C, washed with PBS, and incubated with secondary antibodies and 4,6-diamidino-2-phenylindole for an additional 2 h at room temperature. Coverslips were washed and mounted using ProLong antifade (Molecular Probes). All images were acquired on a DeltaVision RT system (Applied Precision) with a ×100/1.40NA UPlanSApo objective (Olympus) using SoftWorx software. Images are maximum intensity projections of deconvolved stacks.
For quantification of immunostaining, all images of similarly stained experiments were acquired with identical illumination settings and analysed using ImageJ. An ImageJ macro was designed to threshold and select all centromeres and all chromosomes areas (excluding centromeres), using the 4,6-diamidino-2-phenylindole and ACA channels. The convolve filter was first applied to the ACA channel and the threshold selection increased by 1 pixel (to ensure complete kinetochore selection). This was used to calculate the relative mean kinetochore intensity of Mad2, Mps1 and Hec1 ([centromere–chromosome arm intensity (Mad2/Mps1/Hec1)]/[centromere–chromosome arm intensity (ACA)]). For centromere localization of AurB/pAurB, a true background outside the cell was used to subtract from the centromere intensity (and centromere areas were enlarged by 3 pixels to incorporate the more diffuse Aurora B staining).
For quantification of cyclin B–mCherry fluorescence, ImageJ was used to calculate the total integrated fluorescence intensity of individual cells at each time point (after background subtraction of all images using a rolling ball radius of 200 pixels).
For all kinetochore intensity quantifications, means and standard deviations were calculated for each individual experiment and then combined using the standard method for non-overlapping data sets.
