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Cell profiling using automated microscopy: HCS/HCA

Published on 5 August 2022
Research and development activity
Leader Emmanuelle Soleilhac

The main aim of the R&D activity of the platform is to develop quantitative and qualitative imaging tools based on High Content Screening technology (HCS) for the discovery of new bioactive compounds by robotic screening or for their validation and characterization by an in-depth cellular profiling.

High Content Screening is a fluorescent automated imaging technology allowing the screening of compounds on fixed or live cells. It allows the rapid imaging of many cells on a given experimental condition and the automatic extraction of huge sets of quantitative and qualitative features at the cell level (count, area, intensity, form… for a number of objects like cells, nuclei, vesicles, fibers…).

Since 2007, our goal is to develop a cellular profiling strategy in order to speed up the validation and characterization step of the bioactive compounds (Hits) selected by robotic screening on our platform. For that purpose, different cell-based assays were set up in 96-well plates and allow the investigation of the compounds effect on intracellular components (Figure 1). For each experimental test, a specific automated image acquisition protocol is developed with a view to routine applications. Huge data sets are produced and require the development and integration of computerized object segmentation methods, extraction of specific descriptors, pattern recognition and statistical modelling.

Figure 1: Cellular profiling strategy using HCS/HCA

Furthermore, in the context of Gen&Chem scientific projects, we apply this cell profiling strategy to the study and the understanding of the regulation mechanisms of innate immunity and inflammation. More especially our goal is to discover new interesting chemical probes targeting DeUBiquitinating enzymes (DUBs) which represent major actors of these cellular functions. Cell-based assays have been developed to quantify with accuracy either the TNFα-induced nuclear translocation of NF-κB or autophagy (Figure 2). These assays were used for the screening of a set of known chemical compounds in dose-response and for the study of the genetic extinction (shRNA) of few DUBs (ongoing projects).

Figure 2: Cell-based assay for autophagy quantification.
HeLa cells expressing LC3, a specific autophagosome marker, coupled to GFP were stained with Hoechst for nuclei recognition. Images of Hoechst and GFP channels were acquired with INCell Analyzer 1000 (20x obj). Then using INCell Investigator software, images were processed leading to the extraction of quantitative and qualitative features as the number of cells, the intensity of each staining, the shape or the area of nuclei, cells and autophagosomes.

A statistical collaborative project is conducted with Pr Robert Nadon (McGill University, Montreal, Quebec) with the aim to develop novel statistical methods to determine reliable data and to appropriately analyze the biological effects of compounds. These methods should allow easier conversion of the visible biological information (microtubule depolymerisation, cell death, damaged mitochondria etc.) as a series of statistically quantitative descriptors usable for the description of pharmacological signatures of compounds.

Skills and collaborations: Few examples.

• Cell-based assays for HCS/HCA applications.