Development and validation of primary screening assay for identification of cell cycle modulators.

Introduction

High content screening (HCS) is a method that uses automatic microscopy and image analysis techniques to extract multiple phenotypically relevant measurements at cellular level. The method is particularly useful approach for screening of potential cell cycle modulators. We exploited HCS for cell cycle analysis by using HeLa cells stably expressing FUCCI (Fluorescent Ubiquitination-based Cell-Cycle Indicator) probe.

The FUCCI probe was generated by fusing red and green fluorescent proteins with ubiquitination domains of Cdt1 and Geminin respectively. As a means of tracking cell cycle progression FUCCI exploits cyclical expression and degradation of the ubiquitination oscillators Cdt1 and Geminin to specifically mark cell cycle phases in living cells. Cell cycle perturbation can be evaluated by measuring mean fluorescence intensity of Cdt1-Red and Geminin-Green in single cell and eventually based on fluorescence intensity range cells can be classified as one of G1, G1/S, S/G2/M or M cell cycle phase.

Materials/methods

We used FUCCI probe to set up robust assay for efficient screening of large library of compounds. Assay was designed in 1536 well format for full automatization on robotic station which has liquid dispenser, incubators and Yokogawa CV7000 automated microscope connected. This means that cell plating, addition of compounds, cytotoxicity measurement and high content microscopy is processed by robotic system. Data analysis involves exporting images to Columbus image analysis system and process final results in Dotmatics and Tibco Spotfire software. The assay was validated on HeLa and U2OS cells expressing FUCCI probe by screening 1280 compounds from Lopac and Prestwick libraries.

Results and conclusions

Full workflow of assay development and validation will be demonstrated. The results we obtained shows that our robotic station is fully capable for primary screen for large number of compounds and that developed assay is simple to set up, robust, sensitive and inexpensive.

This work was supported by grants LF_2015_031 and by the National Sustainability Programme (LO1304).