Monitoring Cell Death in Real‐Time/Time‐Lapse Studies

Abstract

Personalised medicine, chronic exposure drug toxicity and environmental exposure amongst others have created the demand for in vitro assays which are more physiologically relevant. One aspect of this may be to run assays for many days and monitor them in real‐time or time‐lapse mode. Therein, it can be useful to assess the general cell survival or perhaps a specific cell death pathway in the context of the treatment or insult under observation.Depending on the sample under analysis, to date, this can be done by either marking the cells that remain functionally competent or measure release of ATP at a bulk level. It would be preferable if, conversely, only dead/damaged/apoptotic cells were marked in a binary manner, with a convenient and spectrally separated emission signature and with specificity for a predictable intracellular target such as gDNA, and cell‐by‐cell.To explore this need a novel far‐red DNA binding viability probe, DRAQ7, has been developed. It has been shown to have undetectable toxicity in long‐term/real‐time cell based assays as validated in recent publications (Akagi et al., 2013, Marciniak et al., 2013, Wang et al., 2015, Liang et al., 2015, and internal data) including ultra‐sensitive bioassays for DNA intercalators, ex‐plant tissue culture and nano‐particle toxicity, time‐lapse apoptosis assays and importantly in the presence of toxicants/anti‐cancer compounds.Thus, cells can be exposed to it at any stage of an assay to permit real‐time monitoring of loss of membrane integrity (in apoptosis, death). Being DNA specific it allows monitoring cell‐by‐cell while its spectral properties mean it can be incorporated into multi‐colour flow cytometry experiments, or with Hoechst 33342 or CyTRAK Orange (for simple cell health assays) or with mitochondrial membrane potential probes such as TMRM.DRAQ7 is truly cell impermeant yet retains the DNA targeting and far‐red fluorescence of the parent DRAQ chromophore. Accordingly, it likewise can be used on HCS imaging platforms, fluorescence microscopes, cytometers and sorters. Its spectral properties (long wavelength) are particularly suited to penetrative imaging of multi‐cellular structures and thick ex‐plant tissue sections.The presentation will focus on the deployment and application of DRAQ7 to demanding real‐time and 3D micro‐tissue assays including patient‐derived samples that enable strategies for personalised medicine, underpinned by core performance data that define its unique properties.Support or Funding InformationThis submission is funded by my employer, Biostatus Limited.