CyTRAK Orange - uncovering a drug MoA

The DNA and cytoplasmic dye CyTRAK Orange™ has aided comprehensive studies to elucidate the mechanism of action of a candidate therapeutic for cardiac complications of Chagas' Disease. Tc24-C4 is a modified recombinant protein vaccine derived from a calcium binding protein located in the flagellum of Trypanosoma cruzi.

Interestingly it was clear from the data that the w.t. Tc24 protein is expressed differentially in the different host stages of the parasite's life cycle and that it is expressed in the inner side of the flaggelar membrane.  This explains why Tc24-C4 does not offer protection against infection but operates as a vaccine to generate T cell mediated cell (CTL) killing of infected host cells and thereby ameliorate the effects of infection.

In one experiment cells were infected with T. cruzi and the progression from the infectious extracellular flagellate trypomastigote stage through to the intracellular, fully non-flagellate amastigote stage.  The host cells and parasites were stained with anti-Tc24 to detect stage-specific protein expression, Hoechst to label the nuclei, CyTRAK Orange™ to label the DNA of parasites and cells and DNA and cytoplasm of the host cells.  From this combination of three reagents it was possible to visualise the progress of the infection, from trypomastigote to amastigote and the latter's multiple divisions alongside the progressive loss of expression of Tc24.

Short of including its ability to cross the plasma membrane this application of CyTRAK Orange™ demonstrates its abilities to label DNA (here of host and parasite) and cytoplasmic compartment while being spectrally compatible with Hoechst and Alexa Fluor/GFP/FITC (and red-excited fluors - not used here).

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Reference:

Versteeg L, Adhikari R, Poveda C, Villar-Mondragon MJ, Jones KM, Hotez PJ, et al. (2021) Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi. PLoS Negl Trop Dis 15(9): e0009689.

Read a recent review on CYTRAK Orange™.

Automated dysplasia analysis in MDS

Towards a better and more robust assessment of dyserythropoiesis in myelodysplastic syndrome (MDS) clinical scientists at Aarhus University Hospital have built on the community's development of multicolour flow cytometry in recent years.  They have utilised imaging flow cytometry to gain both immunophenotypic and morphological information on a cell-by-cell basis.

Frozen bone marrow MNCs were thawed and treated with DNase. For exclusion of dead cells Zombie Violet™ Fixable Viability dye (Biolegend) was then added. Following a wash, an antibody panel targeted against CD235a, CD105 PE-CF594, CD71, CD117 and CD45 was added to the cells. Finally, for the morphological analysis, in addition to bright field (BF), DRAQ5™ (for DNA; 1.25 µM) and Thiazole Orange (TO; for RNA; 0.25 µg/ml) were added to the cells and analysis undertaken. The latter reagents are a well-tested combination from early work to unpick the normal course of erythropoiesis (by K. McGrath and colleagues, Rochester, NY).

To better perform the sample analysis on the ImagestreamX Imaging Flow Cytometer, novel machine learning tools were applied to enable the separation of true binucleated events from doublets, aided by a combination of DRAQ5, BF and CD235a.

The authors show proof-of-concept for the detailed analysis of dysplasia in erythroid differentiation towards more definitive diagnosis and monitoring of MDS, most importantly perhaps reducing the intra-operator variability and alleviating the issues related with cytopenic samples.  

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Reference:

Rosenberg C, et al. Cytometry. 2021;100:554–567

Further reading:

One earlier report cited in this paper, used CyTRAK Orange™, for multicolour flow cytometry for investigation of MDS, enabling a no-lyse approach to the sample analysis.

At the time of writing (Sept. 2021) DRAQ5 has been cited alongside the Imagestream imaging flow cytometer in ca. 500 peer-reviewed articles.

Designing a High Content Screen?

If you are working on the design of a high content screen that you think could utilise a BioStatus product then please contact us to discuss your requirements.  We will offer you scientific advice and take advantage of the knowledge gained through citations of our products in more than 10,000 peer-reviewed journal articles.

BioStatus products have been utilised in high content screens for 2D and 3D assay systems by major pharmaceutical companies, research institutes and academic laboratories around the globe for more than 18 years.  The first verified use of DRAQ5 in a screen was in 2003 at Astra Zeneca's Charnwood site!

BioStatus personnel have participated actively in high content screening scientific conferences throughout its history: SBS (later SLAS), CHI's High Content Analysis, ELRIG's "Drug Discovery" and "Pharmaceutical Flow Cytometry and Imaging", SBI2's "High Content" annual conference,  workshops at ICCB and Experimental Biology amongst many others and contributed to a number of training courses.

First, we want to make sure that you use the right product and we will communicate with you the details for its place in your protocol and how it might be analysed on your high content platform.  If we don't think it's right for you we will tell you - your success is always the priority for us.

Once you've tested it at bench-scale and are confident it is transferable to your high content platform we can together consider the bulk quantity required and the discount that would apply along with the best package/presentation/schedule.

Significant discount is available for bulk purchases of multiple millilitre single order purchases and for repeat, call-off and standing orders - we are flexible to your needs.

Once everything is agreed we will make sure that you have your shipment of a single lot number of product well in advance of the start and, if requested, we would be delighted to supply a test vial of that same lot number for your pilot experiment along with your purchase (as a matter of best practice).

Contact us - let's start the discussion!

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Imaging hypoxia in 2D & 3D

A recent paper explores the use of fluorogenic dyes to image the hypoxic response of cells via CYP450 enzymes. This is the first paper citing the use of the far-red fluorescent reporter HypoxiTRAK™ (BioStatus Limited), published by research groups at the Departments of Chemistry and Oncology, University of Oxford. 

The authors describe the development and performance of bioreductive azide dyes. HypoxiTRAK™ is BioStatus's commercially available probe for the detection of a functional reductive potential or response in cells experiencing hypoxic stress, and used here to validate the performance of the authors' experimental molecules.  

References: 

O’Connor, Liam J., et al. "CYP450 enzymes effect oxygen-dependent reduction of azide-based fluorogenic dyes." ACS central science 3.1 (2017): 20-30. (Fig. S2).

UPDATE: HypoxiTRAK™ is also compatible with real-time/time-lapse monitoring of the hypoxic experience of cells in both 2D and 3D microtissues and spheroids, over several days.  The latest paper from the expert lab of Prof. Paul A. Johnston, UPMC (Pittsburgh) can be found here:

Close, David A., and Paul A. Johnston. "Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines." SLAS Discovery 27.1 (2022): 39-54.

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Related products:

DRAQ9™ has been demonstrated for the tracking of cell migration and the monitoring of spheroid growth and integrity: 

Poster presentation, SLAS 2020

DRAQ7™ has been widely used in 3D tumour microtissues to track cell death over time.  Read recent blog reports:

Cancer Drug Screening in PDTOs - key parameters

Real-Time Cell Health Monitoring in 2D/3D with DRAQ7