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Authors

Siang-Boon Koha, Patrice Mascalchia,b, Esther Rodrigueza, Yao Lina,c, Duncan I. Jodrella, Frances M. Richardsa and Scott K. Lyonsa,d

Affiliations

a Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, United Kingdom
b Bordeaux Imaging Center, UMS 3420 CNRS-Université de Bordeaux-US4 INSERM, Pôle d’imagerie photonique, Bordeaux F-33000, France
c College of Life Sciences, Fujian Normal University, Fujian 350117, P. R. China
d Cold Spring Harbor Laboratory, 1 Bungtown Road, New York 11724, United States

Application Area

Cell Biology

General Fluorescence Microscopy

Abstract

The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput and quantitative robustness. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells, using an all-in-one expression cassette FastFUCCI. The approach obviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2-M but eventually advanced through mitosis into interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently reentered S/G2. Accordingly, we showed evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrated the utility of the FastFUCCI assay in quantitating spatiotemporal dynamics and identified its potential in preclinical drug development. Journal of Cell Science • Advance article

Extract

With the Nikon microscope, red and green fluorescence were captured using a pE-300white CoolLED filtered by Nikon FITC B-2E/C and TRITC G-2E/C filter cubes, respectively. With the Zeiss microscope, they were captured using a pE-4000 CoolLED filtered by Zeiss 38HE and 43HE filter cubes, respectively. To achieve optimal image resolution without excessive illumination, a binning factor of 2 by 2 for the sCMOS camera and 5 by 5 for the CCD camera was applied prior to imaging. Throughout each experiment, the ambient conditions of the imaging platform (e.g. external lighting) were maintained to minimise variations in optical resolution and illumination

Product Associated Features

pE-300white

Product Type

pE-300white

Journal

Journal of Cell Science

Year of Publication

2016

Country of Publication

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