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bioRxiv · 10.1101/2020.04.21.052803

Synchronization of human retinal pigment ephitilial-1 (RPE-1) cells in mitosis

Scott, S. J.·Suvarna, K.·D'Avino, P. P.
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Abstract

Human retinal pigment ephitilial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4/6 inhibitor PD 0332991 (palbociclib) and the microtubule depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Furthermore, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.

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Scott, S. J., Suvarna, K., D'Avino, P. P.. 2020-04-21. Synchronization of human retinal pigment ephitilial-1 (RPE-1) cells in mitosis. https://doi.org/10.1101/2020.04.21.052803

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