Computational protocol: Cenp-meta is required for sustained spindle checkpoint

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Protocol publication

[…] In vivo imaging of living neuroblasts of third instar larval brains were carried out as described previously (). Brains were imaged in a temperature-controlled room set at 21°C with a spinning disk confocal head (Ultraview; PerkinElmer) mounted on an inverted microscope (DMI6000; Leica) with a Zeiss 100× NA 1.4 lens and a camera (QuantEM 512SC; Photometrics), all piloted by MetaMorph 7 (MDS Analytical Technologies). At 20-seconds intervals, a z series of images consisting of seven 1-µm steps was acquired with 1× binning. Confocal video frames are maximum intensity projections. Time-lapse image series were converted into videos with ImageJ software (National Institutes of Health), and still images were processed using Photoshop (Adobe). NEB was defined as when the RFP-Spc25 or RFP- or GFP-Rod signal began to be visible on kinetochores. Anaphase onset was defined as the moment sister kinetochores (marked with either Spc25 or Rod) began to separate. GFP-cyclin B degradation dynamics was measured as described previously (). Briefly, neuroblasts expressing one copy of GFP-cyclin B and RFP-Rod were filmed as above. GFP fluorescence in each z section was quantified for the whole cell and for a central region containing the kinetochores and most of the spindle. This gave a more robust measurement of the OCBD because spindle-associated cyclin B is the first to be degraded during metaphase (). The signal was adjusted for background and for bleaching relative to the signal of a neighboring non-mitotic cell (assumed to be constant). In the graphs for , the signal levels for the whole cell are displayed as normalized signal relative to the maximal intensity measured for the cell. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Xenopus laevis, Homo sapiens, Drosophila melanogaster
Diseases Heart Arrest
Chemicals Colchicine