At the recent ELRIG Research & Innovation 2017 conference at Homerton College, Cambridge there was an excellent keynote address by Richard Eglen and an associated parallel session on the demand for cell-based assays to be performed in 3-D for improved physiological relevance. Alongside this, we’ve received a lot of enquiries about the use of the far-red viability probe DRAQ7™ to monitor cell health in real-time and 3-D assays. Here, we try to review recent relevant papers as well as assessing the suitability of DRAQ7™ for this emerging technology area.
The possibility of real-time cell health assays was usefully addressed by Zhao et al., in 2010. They attempted to use propidium iodide (PI) as a real-time reporter of cell health however it is clear that, despite remaining viable, cells experienced a profound increase in overall fluorescence, initially seen at the plasma membrane, then after 8 hours in cytoplasmic granules (pointing to an active endocytotic uptake) and progressively into nucleoli. This phenomenon is mirrored in flow cytometry when, in samples stained with PI, intact (i.e. nominally negative) cells accumulate a fluorescence signal interfering with the initial s:n ratio between intact and compromised cells, observed as a right-ward drift of the negative population on an intensity plot.
i) It should be noted that this undesirable staining of cells by PI may be undetectable in low magnification cell imaging cytometry platforms and is only seen in higher power microscopes or microscope-based systems.
ii) PI is excited at 488 nm. This blue light excitation is implicated in the autofluorescence emanating from naturally occurring ring-like molecules - NAD(P)H, aromatic amino acids, and so on. Such autofluorescence may become even more problematic in thicker and more complex samples, adding to out-of-focus light and increasing background.
In later work, the same laboratories (Akagi et al, 2013) embarked on a detailed investigation of the possible application of a new far-red viability probe DRAQ7™ to address the evident drawbacks of PI. They found no evidence of DRAQ7™ entering intact cells over several days even at high concentrations, which was further undetectable using ultra-sensitive bioassays for DNA binding compounds and consequent DNA damage signalling. Similarly, DRAQ7™ caused no modulation of the effects of various reference cytotoxic compounds compared to controls.
Usefully, DRAQ7™ avoids autofluorescence as it is excited by red light at 635 nm (or 594 nm) while its far-red fluorescence makes it spectrally compatible with Hoechst 33342, CFP, GFP, YFP, DsRed and TMRM for example.
Subsequently, DRAQ7™ has been utilised in a variety of 2-D time-lapse / real-time applications to monitor for cell health. Examples include: cell health in pancreas tissue-culture (7 day assay; Marciniak et al., 2013), kinetic study of side population analysis (Smith et al., 2013) nano-particle toxicity (40+ h; Ware et al., 2014), mitochondrially-regulated apoptosis (72 h; Liang et al., 2015), cell number expansion versus cell death in tumor inhibition (72 h; Stratikopoulos et al., 2015), host-directed therapy to eliminate Legionella (72 h; Speir et al., 2016) and monitoring cell death in magnetic hyperthermia (24h; Blanco-Andujar et al., 2016).
Extending this, DRAQ7™ has been applied to real-time assays of 3-D spheroids and micro-tissues to better reflect physiological conditions. In one example DRAQ7™ has been used to assess effectiveness of cytotoxic compounds against patient-derived glioblastoma tumour spheroids. This work is described in a recent white paper jointly generated with Imagen Biotech.
Most recently, investigations of the homeostasis pathway as a possible therapeutic route for mTORC1 hyperactive tumors utilised DRAQ7™ as the unified cell viability readout in flow cytometry and imaging of 3-D spheroids and their subsequent outgrowth (Dunlop et al, 2017).
DRAQ7™ has been used on conventional microscopes, popular high content imaging platforms (Incucyte ZOOM, Arrayscan, Celigo S, Operetta, Opera, ImageXpress) and most flow cytometers and cell sorters. As a DNA binding viability probe, in image-based or flow cytometric assays, DRAQ7™ reports death event-by-event and dynamically to 100%, not being limited by indirect signals such as residual ATP from dead cells. Furthermore, such image-based cellular-level analysis can offer further textural information beyond that achieved by "wellular" analysis by plate reader.
In practice, DRAQ7™ can be applied to long-term culture at 1-1.5 µM for reliable long-term reporting of cell permeabilization (death), and persists stably in culture medium without suffering chemical degradation or photo-bleaching. It will only need replacement when any medium exchange is required.
Akagi, J., Kordon, M., Zhao, H., et al. (2013). Real-time cell viability assays using a new anthracycline derivative DRAQ7. Cytometry Part A 83, 227-234.
Blanco-Andujar, C., Ortega, D., Southern, P., et al. (2016). Real-time tracking of delayed-onset cellular apoptosis induced by intracellular magnetic hyperthermia. Nanomedicine, 11(2), pp.121-136.
Dunlop, E.A., Johnson, C.E., Wiltshire, M., et al. Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells. Oncotarget. Published online March 15, 2017. doi: 10.18632/oncotarget.16232
Liang, J. R., Martinez, A., Lane, J. D., et al. (2015). USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death. EMBO reports e201439820.
Marciniak, A., Selck, C., Friedrich, B., & Speier, S. (2013). Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ. PLoS One 8 (11) e78706.
Smith, P. J., Wiltshire, M., Chappell, et al. (2013) Kinetic analysis of intracellular Hoechst 33342—DNA interactions by flow cytometry: Misinterpretation of side population status? Cytometry Part A 83, 161-169.
Speir, M., Lawlor, K. E., Glaser, S. P., et al. (2016). Eliminating Legionella by inhibiting BCL-XL to induce macrophage apoptosis. Nature Microbiology, 1, 15034.
Stratikopoulos, E. E., Dendy, M., Szabolcs, M., et al. (2015). Kinase and BET inhibitors together clamp inhibition of PI3K signaling and overcome resistance to therapy. Cancer cell, 27(6), 837-851.
Ware, M. J., Godin, B., Singh, N., et al. (2014). Analysis of the influence of cell heterogeneity on nanoparticle dose response. ACS nano, 8(7), 6693-6700.
Zhao, H., Oczos, J., Janowski, P., et al. (2010). Rationale for the real‐time and dynamic cell death assays using propidium iodide. Cytometry Part A, 77(4), pp.399-405.
DRAQ7™, a unique far-red viability dye used as a measurement of cell viability in patient derived glioblastoma stem cells. Imagen Biotech & BioStatus, February 2015.Real-time cell health monitoring, cell-by-cell, in 2D and 3D in the far-red. BioStatus, March 2016.