Figure 2.

Cdc20 release assay

(A) Schematic of Cdc20 release assay (MGY1293). The Cdc20 release assay monitors the effect of attached kinetochores with reduced tension on the timing of anaphase onset by treating cells with Taxol only after spindle assembly is complete. This isolates the effect of low tension on attached kinetochores. Cells are first synchronized in G1 using alpha-factor, and then released and subsequently arrested in metaphase by Cdc20 depletion. This metaphase arrest allows for complete spindle formation and establishment of bipolar attachments with unperturbed microtubule dynamics before Taxol treatment. Cells are then released into media containing Taxol in DMSO, or only DMSO as a control, and samples are withdrawn and fixed over the next 90 min to monitor anaphase onset via DAPI staining of DNA (blue spots).

(B) Results from the Cdc20 release assay where symbols represent the mean ± SEM from 3 different experiments. Percent anaphase cells at each time point is calculated by the total number of anaphase large budded cells / (anaphase large budded cells + metaphase cells) ∗ 100. For each timepoint 100–200 cells were scored per experiment.

(C) If scaling up the volumes in this assay, the amount of methionine used in the metaphase arrest can affect the speed and synchronicity of metaphase release into anaphase. Keep the ratio of total methionine (i.e., concentration ∗ volume) to cell number similar to the original Cdc20 release volumes and adjust accordingly if needed.