HCS Applications Targeting Generic Cellular Parameters and Morphology

In contrast to target-based approaches, assays which recapture generic cellular parameters deliver in-depth results with regard to the mode of action of screening compounds.

An elegant HCS approach targeting cellular communication was recently performed at Aventis Pharmaceuticals, USA, whereby an assay was set up to identify compounds that block gap junctions between cells. Cells were labeled with the dye calcein and dispensed with unlabeled cells into 384-well plates. The assay was run as a live cell imaging assay in order to prevent dye leakage which occurs when cells are fixed.

The primary screen was run with 486 000 test compounds, from which 1515 primary hits were identified; among these were 53 compounds that produced concentration-response curves. As the read time per plate was 43 min, in order to prevent passive leakage of the calcein dye from the cells, the consequent throughput was limited to only 13 assay plates per day. This low throughput was partially compensated by the application of four test compounds per well.

This screening campaign highlighted one of the benefits of HCS, namely that it also permits HTS for those targets not amenable to primary screening by using more conventional assays such as radioactive metabolite transfer or dye transfer after microinjection [106].

Cellular assays with multiparametric readouts are desired when a multifaceted cellular phenomenon such as apoptosis is studied. Such an assay was set up to analyze three parameters that are cellular hallmarks ofapoptosis, namely caspase-3 activation, nuclear condensation, and mitochondrial membrane potential. Using two tumor cell lines (HeLa and u937 cells), standard cytotoxic agents were applied and the three apoptosis markers measured simultaneously using an antibody against activated caspase-3, the Hoechst 33342 dye for nuclear staining, and chloromethyl-X-rosmaine to measure mitochondrial membrane potential.

The value of this assay was demonstrated by the actions of the applied drugs, which induced caspase-3 activation and nuclear condensation, but reduced the mitochondrial membrane potential. This multiplexed HCS approach combined biochemical (caspase-3 activation) and morphological (nuclear condensation) information in one assay, thus eliminating the need for different, laborious, single-point assays [107].

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