Description

4D image series of HeLa cells cotransfected with CENP-B–GFP (centromeric protein B) and either control (CIL:13368) or Bod1 siRNA (this image). Bod1 is a protein that associates wtih a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. .

Technical Details

48 h after transfection, cells were split onto 40-mm-diameter glass coverslips (Bioptechs), cultured overnight, and transferred to CO2 independent media with supplements. Cells were maintained at 37°C using an FCS2 chamber in conjunction with an objective heater (Bioptechs). Images were acquired on a restoration microscope (DeltaVision Spectris; Applied Precision) with a 100× 1.35 NA objective and a cooled charge-coupled device camera (CoolSNAP HQ; Roper Scientific). SoftWorx software (Applied Precision) was used for image analysis. Datasets were deconvolved using the constrained iterative algorithm (Swedlow et al., 1997; Wallace et al., 2001) using SoftWorx software. Time courses were presented as maximum intensity projections of deconvolved three-dimentional datasets. Images were loaded into Photoshop (Adobe) or OMERO (http://openmicroscopy.org) and adjusted for display. Image is part of Fig 4B in JCB vol. 179 no. 2 187-197