Cell-based Imaging and Screening Applications

Oncotherics is advancing novel hypoxia-activated pro-drugs as therapeutics in a range of cancers (such as pancreatic and prostate) and has recently signed a memorandum of understanding (MoU) with a leading cancer centre to progress the work. Oncotherics' goal is that these targeted therapies would be a part in the improved survival and standard of care for patients with refractory cancers. BioSuspensions has an exciting patented technology for the effective suspension of insoluble drugs, a major challenge to their presentation to patients, even for the most basic of medicines in the case of dysphagia. There are opportunities to license the technology and to partner on formulation development for the extension of drug patent life, realisation of previously insoluble molecules and access to new market segments due to improved formulation. Altogether, we at the Biostatus group are proud to have advanced both of these British inventions alongside those of our original business!

DRAQ5 has been applied to cell-based screening and can be used to mark the nucleus of a cell (effectively the placeholder for the measurement of each cell) whether this is by fluorescence based imaging or by high-throughput flow cytometry. The staining intensity can give information on DNA content – where a cell is quiescent or actively dividing or whether a treatment has arrested the cell in a particular part of the cell cycle. Imaging of the cell allows morphometric information to be gathered and the size and shape of the nucleus has proved diagnostic, in many assays DRAQ5 permits these measurements. Interestingly, a weak secondary cytoplasmic signal can also be utilised to get the same measurements of the cellular envelope. This turned out to be an unexpected USP for DRAQ5 in the early days of its adoption. As a cell-permeant probe, DRAQ5 can be used in ether fixed- or live-cell endpoint assays and combining this with its DNA specificity and far-red fluorescence have made it the ‘go to’ DNA counterstain for high content screening labs around the globe.

Two striking developments by customers of DRAQ5 have been in very different fields. The first is histopathology where DRAQ5 has been used to replace the workhorse dye hematoxylin to simplify and accelerate the analysis of tissue biopsies to such an extent that real-time intra-operative pathology reporting is achievable. The second development is in the area of fundamental understanding of chromatin folding and packing where DRAQ5 acts as a catalyst to enable labeling of chromatin and thereby make the nuclear material dense enough for high resolution electron microscope tomography. This has already brought into question the textbook understanding of higher order structures of chromatin, that may have been artefacts of sample preparation.

CyTRAK Orange is a close relative of DRAQ5. It is similarly cell permeant but has a little more promiscuous binding to RNA in the cytoplasm to be a convenient single-channel probe for both nuclear and cytoplasmic envelopes. As its name suggests it fluoresces in the orange (peaking at 610 nm to be precise) but is still green fluorescent protein (GFP) compatible, just like DRAQ5. CyTRAK Orange can also be utilised on flow cytometry platforms, sharing the cross-platform attributes of DRAQ5, and it can be elegantly combined with DRAQ7 for a live/dead combination that’s been described recently in the published literature. My sense is that this is an under-rated and under-utilised probe with great value in screening but lacks that spark of critical mass of citations.

We have been busy in new product development.

We recently launched a variant of DRAQ7 in a dropper bottle presentation to make benchside additions of a viability probe easy yet still get the performance DRAQ7 offers – this is called DRAQ7 DROP & GO and only needs 2 drops to be added to a tube of cells a few minutes prior to analysis. I’ve been collaborating with a European core lab and a cytometry manufacturer to exemplify this probe for complex antibody panels where there's presumed to be no space for a viability marker and DRAQ7 enables this, remarkably!

In the same vein, we have developed a completely new probe formulation called DRAQfx.  As the name suggests it is a far-red DNA probe for counterstaining fixed cells and thin tissue sections in immunofluorescence microscopy.  Again, with DRAQfx FIX & GO one simply adds 2 drops to 1 ml of buffer and stains a batch of slides or multi-well plates without the hassle of freeze-thawing of reagents every time.

In an exciting change of direction, we have launched a new probe called DRAQ9 which again is cell permeant and far-red fluorescing but unusually does not label the nucleus, rather it labels structures in the cytoplasm and with extremely low toxicity. This offers new possibilities for cell painting/mosaicing and cell tracking. The early work on this was recently presented by Professor Rachel Errington of Cardiff University at ISAC’s  CYTO 2017 conference in Boston, Massachusetts, USA.