Spheroid / 3D Culture Cell Health Analysis:
Dunlop, E. A., Johnson, C. E., Wiltshire, M., Errington, R. J., & Tee, A. R. (2017). Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells. Oncotarget, 8(30), 48711.
In cancer therapy research, scientists at Cardiff University have used DRAQ7™ to show the significance of discriminating between cytostatic and cytotoxic anti-tumour treatments. Experiments showed these can be uncovered independent of 3-D spheroid diameter where after removal of cytostatic treatment spheroid growth may recover, indicating an "escape" from the intended outcome of the therapy.
In these experiments spheroids were exposed to DRAQ7™ for cell health monitoring for 36h, and with spheroid diameter was the only parameter required to observe post-cytostatic recovery or cytotoxic collapse.
Yu, K. K. H., Taylor, J. T., Pathmanaban, O. N., Youshani, A. S., Beyit, D., Dutko-Gwozdz, J., ... & Bigger, B. W. (2018). High content screening of patient-derived cell lines highlights the potential of non-standard chemotherapeutic agents for the treatment of glioblastoma. PloS one, 13(3), e0193694.
In the search for improved prognoses for glioblastoma patients clinical and academic researchers in Manchester, UK have compared 2D and 3D (neurosphere) assay formats for physiological relevance. Patient-derived cell lines were challenged with a panel of 83 drugs FDA-approved for a variety of other cancers to determine if there was useful cytotoxicity that might point to benefit in a personalised medicine approach to treatment.
The high content screen of cell/neurosphere exposure to a 5-log dilution series of each of the drugs for 96 h, and a final incubation with Hoechst 33342 (to count cells/neurospheres) and DRAQ7™ to determine cell death).
Sigmundsson, K., Ojala, J. R., Öhman, M. K., Österholm, A. M., Moreno-Moral, A., Domogatskaya, A., ... & George, B. (2018). Culturing functional pancreatic islets on α5-laminins and curative transplantation to diabetic mice. Matrix Biology, 70, 5.
This work from Duke-NUS Medical School, Singapore and Karolinska Institute, Stockholm showed the potential for a sustained reversal of Type 1 Diabetes by transplantation of islets allowed to spread onto basement membrane protein α5-laminin coated PDMS membrane.
These islets showed excellent viability and functional capacity over many days. In contrast, islets maintained in suspension culture rapidly developed large hypoxic (MAR+), necrotic cores DRAQ7™+) which were shown to be expelled, as shown in striking video and static images.
Lee, S. W. L., Adriani, G., Ceccarello, E., Pavesi, A., Tan, A. T., Bertoletti, A., ... & Wong, S. C. (2018). characterizing the role of Monocytes in T cell cancer immunotherapy Using a 3D Microfluidic Model. Frontiers in immunology, 9, 416.
To investigate the inhibitory role of monocytes in T cell cancer immunotherapy researchers at A-STAR, Singapore and MIT, USA developed a 3D intrahepatic tumor microenvironment microfluidic model. Importantly they showed that in the equivalent 2D system monocytes did not inhibit T-cell killing of target hepatocytes, suggesting that the 3D model was a superior and more physiologically relevant system.
HepG2 hepatocytes were tagged with GFP, T-cells labelled with Cell Tracker Violet and resulting dead cells monitored by DRAQ7™ in the culture medium, with the three fluorescence signals measured by confocal z-stack fluorescence imaging at 0h and 24h. Dead HepG2 cells were defined as GFP-dim, DRAQ7™+ objects. Monocytes were not labelled.
To investigate the inhibitory role of monocytes in T cell cancer immunotherapy researchers at A-STAR, Singapore and MIT, USA developed a 3D intrahepatic tumor microenvironment microfluidic model. Importantly they showed that in the equivalent 2D system monocytes did not inhibit T-cell killing of target hepatocytes, suggesting that the 3D model was a superior and more physiologically relevant system.
HepG2 hepatocytes were tagged with GFP, T-cells labelled with Cell Tracker Violet and resulting dead cells monitored by DRAQ7™ in the culture medium, with the three fluorescence signals measured by confocal z-stack fluorescence imaging at 0h and 24h. Dead HepG2 cells were defined as GFP-dim, DRAQ7™+ objects. Monocytes were not labelled.
2-D cell Health Assays
Szemes, M., Greenhough, A., Melegh, Z., Malik, S., Yuksel, A., Catchpoole, D., ... & Malik, K. (2018). Wnt signalling drives context-dependent differentiation or proliferation in neuroblastoma. bioRxiv, 236745.
In a study into Wnt and its target genes in neuroblastoma (NB), researchers at the University of Bristol have described transcriptionally distinct features that determine a preference for differentiation or proliferation in response to Wnt signaling.
To study the proliferative response, NB cell lines were monitored in the IncuCyte ZOOM (Essen Biosciences) for 24 hours, in the presence of 1.5 µM DRAQ7™, and following Wnt stimulus, imaged every 2 hours for progress to confluence and for a far-red signal denoting each individual dead cell.
Deo, P., Chow, S. H., Hay, I. D., Kleifeld, O., Costin, A., Elgass, K. D., ... & Lithgow, T. (2018). Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis. PLoS pathogens, 14(3), e1006945.
To study the modulation of the host innate immune system by N. gonorrhoeae, researchers at Monash University, Australia allowed macrophages to ingest outer-membrane vesicles (OMV) rich in a Porin PorB.
In time-lapse imaging experiments they demonstrated that Porin B targets mitochondria and leads to caspase-dependent (apoptotic) cell death, in a time-dependent manner. This was determined by a combination of the observed sequence of collapsing TMRM signal (mitochondrial membrane potential loss), transient Cell Event Caspase 3/7 signal (Cytochrome C release and consequent caspase activation) and finally a persistent far-red viability probe DRAQ7™ signal in the nucleus of each cell over a 48 hour time course, monitored every 30 minutes.
Automated, Multi-Parameter, Kinetic Methods to Quantify Cell Death - Application Note
Brad Larson, Principal Scientist, BioTek Instruments, Inc., Winooski, VT USA. March, 2018
An integrated, automated cell incubation and imaging workflow was used to demonstrate a multi-parametric and kinetic methodology to assess cell death. The BioSpa Live Cell Imaging System consists of the Cytation™5 Cell Imaging Multi-Mode Reader and BioSpa 8 Automated Incubator (Biotek Instruments, Inc.).
In the test system, fibrosarcoma HT1080 cells were treated with a 7-point, 4-fold dilution series of Camptothecin in the presence of TMRE (Abcam), pSIVA-IANDB (Abcam), and DRAQ7™ to detect mitochondrial membrane potential, externally-exposed phosphatidylserine and compromised cell membranes / necrosis respectively. Wells were imaged at 2-hourly intervals for 48 hours for the three chromophore signals and for brightfield to assess cell numbers.
Previous methodologies have positively marked cells based on positive metabolic competence. Conversely, DRAQ7™ labels only membrane-compromised (damaged, dying, dead) cells, its red excitation minimises risk of DNA damage when capturing multiple time-lapse images whilst reliably monitoring viability in real-time, cell-by-cell.
DRAQ7™ offers new dimensions and opportunities for performance of high value phenotypic and in vitro toxicity cell-based assays in drug discovery and development, that can be applied across different platforms including flow cytometry, imaging flow cytometry, fluorescence microscopy and high content imaging platforms.
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