Abstract

High Content Death in the Far-red

Roy Edward, Director, Biostatus Ltd

In drug discovery and immunotherapy research two recent technical developments are demonstrating new value to the process, namely: physiologically-relevant 2-D and 3-D cell-based assays (CBA) and high-throughput flow cytometry (HTFC). In both cases, the identification / exclusion of dead cells is a common or key component of the assay. A purpose-designed viability probe, DRAQ7™ has potent utility in both. In many 2-D/3-D CBA a key background parameter is monitoring of cell health/cell death, more centrally in assays for new oncology therapies and where physiological relevance is enhanced by use of long-term / time-lapse readouts. This monitoring therefore needs to be compatible to existing high content screening platforms, inherently non-toxic yet persistent and be detected without collateral damage or sample destruction that might be caused, for example, by UV exposure or by bulk ATP measurement. Uniquely, this can be achieved with the use of the far-red fluorescing DNA binding viability probe DRAQ7™ as described in recent publications in 2-D, 3-D spheroids and ex-plant tissue culture. In high throughput flow cytometry, as in all flow cytometric procedures, it is highly beneficial to exploit the maximum number of channels with the least amount of channel spillover (which otherwise demands compensation) thereby achieving the depth of phenotyping (i.e. population segmentation), sensitivity and dynamic range. Nonetheless, the exclusion of dead cells from analysis is a common requirement in flow cytometry where the conventional viability probes occlude valuable chromophores options e.g. PI of commonly used R-PE or DAPI of the new Brilliant Violet reagents. Utilising the multi-beam excitation of DRAQ7™ it is uniquely possible to place dead cell events into a virtual channel that, therefore, does not occupy any channel and does not require compensation against other channels. The principle underlying this simple strategy will be described with a working example that release all the channels in a HTFC platform for phenotypic analysis while still permitting dead cell exclusion.