Possible breakthrough in GBM therapy

Is this the breakthrough in treatment of glioblastoma that has been evading us now for decades?

Despite very good and well-justified efforts to challenge, for example, 3-D cultures of patient-derived tumor stem cells with the battalion of approved drugs used in cancer therapy nothing has worked.  The debate has raged.. Is it the failure to reach the tumor? Is it inflammation, despite the BBB leakiness that follows? Is it the dendritic and diffuse nature of the disease? Or, and so on?

Here then is something completely novel: a combinatorial approach based on two small molecule drugs: an anti-epileptic, bumetanide, and an anti-helminthic, mebendazole, that shows real promise in killing glioblastoma (GBM) cells.  The work led by Yehezkel Ben-Ari of Brain Tech, INSERM and start-up Ba-oncomedical (both of Grenoble, F) showed significant reduction in cell hyperactivity and increased cell death with the combination therapy against cell cultures and animal models than that achieved by single agent or DMSO control. 

Amongst the investigations performed, DRAQ7 was used in parallel with caspase-3 detection to monitor apoptotic cell death in 3-D co-cultures of neural cells and GBM tumor-derived cells - by flow cytometry, following tumoroid digestion.

The authors' assert that the combination of a NKCC1 inhibitor (e.g. bumetanide) and a microtubule disruptor (e.g. mebendazole) requires further clinical exploration.

At the very least this research suggests that we need to look much wider in our search for a GBM treatment that gives a useful increase in patient survival. That will entail bold investments in industrial scale biology, AI/ML interrogation and a deeper understanding of the GBM tumor milieu to identify lethal susceptibilities similar to that described in the current work.

Reference:
Bourgeois et al. Dual Targeting of Brain Tumors and Their Environment by Bumetanide and Mebendazole. Cancer Research Journal 2025, Vol. 13, No. 3, pp. 98-112.

DOI:10.11648/j.crj.20251303.12

Discovery - platelets sequester cfDNA

Exciting research published recently in the journal Science sheds new and important light on the presence of cell-free DNA (cfDNA) in the circulation as a potential aid to diagnosis for malignant disease.  

Led by Prof. Bethan Psaila, University of Oxford, the multi-centre team describe the significant presence of cfDNA sequestered in platelets using microscopy and flow cytometry to elucidate and quantify this, and genetic techniques to determine the likely chromosomal origins and the inclusion of DNA from pre-malignant and cancerous lesions.

To detect the presence of platelet dsDNA far-red cell-permeant DNA dye DRAQ5 was chosen.

Current approaches to liquid biopsy sample platelet-depleted plasma, and these findings suggest that a substantial proportion of cfDNA and, therefore, important genetic information contained within platelets is being missed.  Beyond the obvious opportunity to increase sensitivity of early cancer detection, especially in the pursuit of cancers prone to late-diagnosis and found in difficult to access tissues, it may also permit access to circulating cfDNA of fetal origin in maternal blood and new avenues in diseases outside of cancer.

Reference:

Murphy, L., Inchauspé, J., Valenzano, G., Holland, P., Sousos, N., Belnoue-Davis, H. L., ... & Psaila, B. (2025). Platelets sequester extracellular DNA, capturing tumor-derived and free fetal DNA. Science, 389(6761), eadp3971.

Cytokine-induced barrier dysfunction via gut-on-a-chip

A gut-on-chip model that generates human intestinal tubules has been used to demonstrate the detectable effect of cytokine exposure, an effect of inflammation in vivo, that causes complex changes in the intestinal epithelial barrier.  

This cytokine-induced barrier dysfunction was measured by TEER (trans-epithelial electrical resistance), combined compromised cell health (DRAQ7) and cytoskeleton changes (actin) and by lipid signaling profiles (LC-MS/MS).

The work was led by Amy Harms and Thomas Hankemeier at the Leiden Academic Centre for Drug Research, NL.

For the combined analysis of cell health and cytoskeleton alterations, following negative control or cytokine exposure of the gut-on-a-chip intestinal tubules, DRAQ7 was used to detect cell plasma membrane failure (i.e. catastrophic or programmed cell death) during a 30' incubation.  Thereafter, tubules were fixed (3.7% PFA) and then permeabilised with Triton X-100 (0.03%) and then stained with NucBlue Fixed Cell ReadyProbes Reagent (Invitrogen, R37606) (for ALL nuclei) and ActinGreen (Invitrogen, R37110) for cytoskeleton.

Using the stated protocol the data show a clear cytokine dose-related increase in cell death.

This method is additionally important in general use since this describes the use of DRAQ7 to stain nuclei of dead/dying cells and to detect this following fixation and permeabilization steps, opening up a new route for DRAQ7's use, perhaps reliant upon careful titration of fixative and surfactant to moderate the required effect to avoid the undesirable transfer of DRAQ7 from the true dead cells to otherwise DRAQ7-negative fixed and permeabilized cells.

The wider results underpin the physiological response of the in vitro tubules in the OrganoPlate organ-on-a-chip system (Mimetas, b.v.) to the inflammatory insult of cytokine exposure.

Reference:

Morelli, M., Savova, M. V., Queiroz, K., Harms, A. C., & Hankemeier, T. (2025). Cytokine‐Induced Barrier Dysfunction and Lipid Signaling in a Gut‐On‐Chip Model. The FASEB Journal, 39(19), e71059

Improved CDC with IgM CD20

A team at IGM Biosciences Inc., led by Kevin Hart and Bruce Keyt demonstrate the improved performance of an engineered IgM anti-CD20 antibody to achieve complement-dependent cytotoxic cell killing for B-cell lymphoma over IgG alternatives.  Due to the development of resistance to the traditional IgG-based therapies such novel antibody alternatives are required.

The IgM gives better killing rates, over a shorter period and crucially of target cells with lower antigen density and in the presence of complement inhibitory conditions.

To understand the dynamics of cell killing by the respective IgM and IgG therapeutic antibodies, target cells were stained with CellTrace Green ("all cells") and in the presence of DRAQ7 to report the failure of the plasma membrane due to the complement effect.  This was achieved in a disposable hemocytometer chamber slide C-Chip (Incyto) under environmental control and recorded using the Lionheart Fx microscope (Biotek, Inc.).

WHERE TO BUY DRAQ7

Reference:
Hart, Kevin C., Paul R. Hinton, Marigold Manlusoc, Kevin B. Carlin, Samuel Schneider, Maya F. Kotturi, Ramesh Baliga, and Bruce A. Keyt. "An engineered IgM antibody targeting CD20 has enhanced complement-dependent cytotoxicity compared to an IgG." Experimental Hematology (2025): 105250.

DRAQ7 and DRAQ5 in parallel for cell death studies

A team at the Univ of Cologne, led by Manolis Pasparakis, sheds new light on the complexity of RIPK1 and its downstream regulation of cell death and inflammation.  This is shown in the context of dermal inflammation and the dysregulatory effects of mutations at the sites of autophosphorylation and how these sites contribute to the pathogenesis of inflammatory disease.

RIPK1 has been shown to be a key regulator of cell death and inflammation but with little knowledge of downstream effects of mutations on its function.  Dysregulation of RIPK1 has been implicated in the pathology of inflammatory disease when the discrete control of its phosphorylation is lost, making it a useful target for therapeutic inhibition.  The key activation is autophosphorylation, dependent upon two sites, to drive downstream signalling towards apoptosis and necroptosis and ultimately to inflammation.  Different mutations enforced at the phosphorylation sites impose a variety of noticeable changes in the downstream outcomes, for example favouring necroptosis over apoptosis.

The work explored in vivo in mice the impact of mutations on keratinocyte biology and resulting skin lesions, examined by histology and for gene expression patterns.  Immunoblotting studies validated the use of cell death to be a direct correlate for the changes imposed by the mutations to the phosphorylation sites.  From this, extensive in vitro cell-based assays were performed for cell death under different mutations and drug regimes. 

In these latter assays performed on the Incucyte S3 live-cell analysis system (Essen Bioscience), the far-red fluorescing cell-impermeant DNA-binding viability dye DRAQ7 was used to dynamically report cell death.  Elegantly, the related cell-permeant DRAQ5 was used in replicate wells to give a robust control baseline for the total number of cells and therefore to allow cell death to be reported as a percentage of the total.  This means that the readouts for total cells and cell death is from the same fluorescence channel, simplifying instrument set-up while providing capacity for further chromophores, were these deemed necessary.  In this context one might suggest a reporter of caspase activity and/or mitochondrial health to further unpick the cell death mechanism.

Where can I buy DRAQ5 and DRAQ7?

Reference:
Koerner, Lioba, et al. "RIPK1 autophosphorylation at S161 mediates cell death and inflammation." Journal of Experimental Medicine 222.12 (2025): e20250279.

Probing GPX4 as a target to drive ferroptosis

Ferroptosis has become a promising target for cancer therapy.  This regulated non-apoptotic cell death process is described as an iron-dependent lipid peroxidation, specifically necessitating peroxidation of polyunsaturated fatty acid-containing phospholipids. The protective mechanism in healthy cells is for the resulting lipid hydroperoxides to be converted to the corresponding lipid alcohols by glutathione peroxidase 4 (GPX4) and that being able to target this enzyme for inhibition in cancer cells could overcome the suppression of ferroptosis.

A collaboration between groups at AbbVie, Inc. and Stanford University, led by Relja Popovic and Scott J. Dixon respectively, explored the scope for GPX4 therapeutically.

In the course of the studies a sensor of lipid peroxidation (Bodipy 581/591 C11) and DRAQ7 were used in combination to demonstrate that cells undergoing ferroptosis accumulate lipid peroxides and subsequently die, and that this could be modulated under different conditions.

What became clear with the complex investigations by the authors was that the transition from 2-D to 3-D cell culture system reduced the sensitivity of cells to GPX4 inhibition - due to a substitution by monounsaturated fatty acids in the 3D culture from the upregulation of another enzyme (SCD).  Under this 3-D culture condition, it is generally understood to recapitulate better the in vivo condition, though for this case needs to be seen as initial findings and requires further investigation.

GPX4 remains limited in its promise as a target to block suppression of ferroptosis due to these confounding data and without the means currently to specifically target cancer cell GPX4 based on another feature e.g. proliferation rate, tumour cell surface marker, etc.

WHERE TO BUY DRAQ7

Reference:

Park, V. S., Pope, L. E., Ingram, J. P., Alchemy, G. A., Purkal, J. J., Murray, M. B., ... & Popovic, R. (2025). Lipid Composition Alters Ferroptosis Sensitivity. Cancer Research. 
https://doi.org/10.1158/0008-5472.CAN-24-4207

MDS Imaging Flow Cytometry with DRAQ5

Features with MDS need to be better defined to be robust indicators of disease.  Disease experts from Lund University and Skåne Regional Laboratories show that Imaging Flow Cytometry (IFC) can provide the statistical discrimination of cellular features that are cellular hallmarks of MDS.

Notably they used DRAQ5 to trigger cellular events, widely used elsewhere.  However, they describe a new identifier of nuclear condensation utilizing the DRAQ5 signal and the Bright Detail Intensity (BDI) feature in the Imagestream's IDEAS software. DRAQ5-BDI showed that there was a definitive reduction in this feature in disease versus normal bone marrow, consistent with degree of chromatin condensation, evident in megaloblastoid cells.

Moreover, they capitalized on the DRAQ5 signal to determine nuclear aspect ratio and nuclear area features and to identify binucleated cells which were weighted towards euploid rather than double DNA status in MDS samples and the reverse in normal bone marrow, reflecting different cell cycle positions and therefore proliferation rates.

Using fresh samples (neither fixed or freeze-thaw treated) they authors were able to more faithfully preserve the integrity of a range of features to segment cell populations and observe phenotypic staining and cytoplasmic features that were also beneficial in stratifying disease.

Where can I buy DRAQ5?

Reference:
Despoina Violidaki, Olof Axler, Lars Nilsson, Anna Porwit, Mats Ehinger. Translation of the Morphological Hallmarks of Dyserythropoiesis to Objective Morphometric Parameters by Imaging Flow Cytometry. International Journal of Laboratory Hematology. 2025 Jul 29. DOI:10.1111/ijlh.14534

Atherosclerosis research - plaque monitoring and cell biology

Two new papers utilizing DRAQ5 to facilitate high quality cell sorting to downstream single cell RNA sequencing to unpick the molecular backdrop to atherosclerotic plaque cell biology.

In the first of these, from Muredach Reilly's lab at Columbia University, fibroblasts were profiled. Four distinct subpopulations were identified - all of which diminished in number upon onset of experimental atherosclerosis in mice.  Significantly, a CD26+ subpopulation migrated from the "adventitial" or external connective tissue layer in healthy individuals and into the innermost layer ("intima") upon atherosclerosis. Notably, these were found to localize to the fibrous cap of the lesion as determined by a combination of flow cytometry and immunohistochemistry. 

The suggestion is that this subpopulation may stabilize the plaque. It may be early to hypothesize if this is driven by other factors (e.g. unrelated inflammation) or whether it has implications for therapy since CD26 is normally expressed on vascular endothelial cells and is involved in cell adhesion.

In the second article, led by Jacob Bentzon with research teams in Madrid and Aarhus, the use of FDG-PET to monitor plaque progression was explored.  Using the data from transcriptomics, it was seen in a large animal model, pig, that glycolytic enzyme expression was reduced concomitant to FDG-PET signals and plaque regression in all associated cell types. 

This direct correlation to the observed FDG-PET signals indicated that PDG-PET imaging could be valuable in monitoring of atherosclerotic plaques as an aid to clinical management.

Where can I buy DRAQ5?

References:
Bashore AC, Coronel J, Xue C, Zhu LY, Reilly MP. Single-Cell Multimodal Profiling Reveals a Novel CD26+ Fibroblast Subpopulation in Atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology. 2025 Jan 28. DOI:10.1161/ATVBAHA.124.322370

Nogales P, Velasco C, González-Cintado L, Sharysh D, Mota-Cobián A, Izquierdo-Serrano R, Torroja C, del Rio-Aledo D, Morales-Cano D, Mota RA, Benguría A. Atherosclerotic disease activity is associated with glycolytic enzyme expression across multiple cell types and is trackable by FDG-PET. Science translational medicine. 2025 Aug 13;17(811):eado6467.

New knowledge on MS progression

It has proved difficult to predict the likelihood of progression of disease in MS.  New work shows that there is a complex relationship between ependymal cells at the tissue interface with CSF and extrinsic factors and also with cell-intrinsic factors that provide new avenues for prognostic and perhaps therapeutic advantage.  

A team led by Dr Jo Anne Stratton, Asst Prof., Montreal Neurological Institute-Hospital, McGill University has uncovered compelling evidence of a correlation between surface-in gradient of tissue damage and disease progression/severity.  Similarly, these ependymal cells (specialised glia) are highly susceptible to modification by IFN𝞬 that results in both changed protein expression and direct morphological changes that predict worse surface-in gradients of disease.

One part of this wide-ranging study was single nucleus RNA sequencing.  Reserved aliquots of isolated nuclei were assessed for quality / blebbing before the downstream processing.  Interestingly, nucleus quality was determined by direct visual microscopic analysis of morphology and counting by flow cytometer; nuclei being labelled with DRAQ5 to achieve analysis on both platforms. 

Where can I buy DRAQ5?

Reference:
Groh, Adam MR, et al. "An MRI-informed histo-molecular analysis implicates ependymal cells in the pathogenesis of periventricular pathology in multiple sclerosis." bioRxiv (2025): 2025-01. DOI:10.1101/2025.01.14.633055

New options for ALS/FTD therapies

A team lead by scientists at Thomas Jefferson University have demonstrated a new opportunity for intervening in ALS/FTD. 

They worked on the hypothesis that aberrant arginine-rich dipeptide repeat (R-DPR) proteins are a key toxicity, aggregating with RNA binding proteins. They demonstrated that it was possible to disrupt the aggregation, though initially at the cost of the function of beneficial liquid-liquid phase separations that occur in stress granules and nucleoli, for example.  Modification of the active protein Kapβ2 to be deficient for the nuclear localisation signal (NLS) enabled them, in vitro, to inhibit R-DPR toxicity without side-effect.

In one demonstration of this, neurones were exposed to combinations of an R-DPR with and without NLS-deficient Kapβ2 in the presence of DRAQ7 as a real-time reporter of toxicity resulting in cell death under the different treatments.  

DRAQ7 again displayed its own ultra-low toxicity over long time-courses (here 18h), enabling such ground-breaking work. Where to buy DRAQ7

Reference:

Kim, K.M., Girdhar, A., Cicardi, M.E. et al. NLS-binding deficient Kapβ2 reduces neurotoxicity via selective interaction with C9orf72-ALS/FTD dipeptide repeats. Commun Biol 8, 2 (2025).  DOI:10.1038/s42003-024-07412-x

3D platform to study EndMT under shear stress

Endothelial to Mesenchymal Transition (EndMT) is a well-understood in development but not in the adult.  This may be important in development of disease or progression through invasion, as in metastasis.  A laboratory at the University of Binghamton, NY has developed a 3D fluidic device that permits the application of shear stress to endothelial cells to determine if this is a driver of EndMT.

To test this required measurement of major cell compartments, cell boundaries and their cell interactions - for example, tight junctions.  DRAQ5 was used to counterstain the cell nucleus at the end of the shear stress period, and this necessitated permeating the collagen matrix utilised and which was tuned to mimic the stiffness of blood vessels.  Using DRAQ5 and fluorescently-tagged phalloidin it was possible to measure the shape index (SI) and orientation angle (OA) under different shear stress conditions, amongst the measurements undertaken to determine the different states of cells under shear stress. 

Where to buy DRAQ5

Reference:

Mina, S. G., Wang, W., Cao, Q., Huang, P., Murray, B. T., & Mahler, G. J. (2016). Shear stress magnitude and transforming growth factor-βeta 1 regulate endothelial to mesenchymal transformation in a three-dimensional culture microfluidic device. RSC Advances, 6(88), 85457-85467.

Imaging Hypoxia in TNBC 3D Microtissues

3D microtissues are now well-established as an improved in vitro recapitulation of tumor and the likely response or resistance to therapeutic interventions.  To that end, a lab at the University of Saskatchewan, Saskatoon, has developed a model micro-tissue platform for triple-negative breast cancer cell lines (TNBC) and having the feature that these microtissues are free-floating in a collagen-based hydrogel as a defined extracellular matrix and to perhaps better establish and in vitro tumor microenvironment.

In one measure of the changes over time during growth of microtissues they were monitored longitudinally for emergence of hypoxic regions from day 7 to 21.  This was achieved non-destructively using HypoxiTRAK™, simply added to culture medium and providing a direct fluorescence readout throughout the time-course.  As expected, these microtissues did show increases in hypoxic microenvironments over the measured period.

WHERE TO BUY HypoxiTRAK

Reference:
Daneshvar BN, Bosso M, Greene R, Dzikowski T, Johannson M, Gagnon A, Chamberlain MD.  A novel in vitro 3D cancer model based on modular tissue engineering approach. bioRxiv preprint, March 1 2025.  DOI:/10.1101/2025.02.25.64023

Screening Concept Aims to Aid Diagnosis for VUS

Fascinating new research headed by a team in Utrecht has applied the strategies now aiding drug discovery to inform and direct clinical investigations of variants of unknown significance (VUS) that are a product of the genetic revolution in medical diagnosis.

With the emergence of VUS that result from their detection by DNA sequencing but which may nonetheless confound a diagnosis or, for example, genetic counselling it has been, thus far, impractical and uneconomical to consider the enormous battalion of tests that might be required to provide a definitive diagnosis, with no certainty of an effective outcome.

They authors of this new work demonstrate that by creating a screening approach they can provide a morphological- and organelle-based feature set for cluster analysis to better direct the clinical investigations for patients with VUS.

They chose the Imagestream imaging flow cytometer to meet statistical sampling demands and to collect image information, from both the assay-designed and inherent image parameters.

Imaging Flow Cytometry and DRAQ5 have long been combined as a solid foundation for assays (500+ co-citations) and this work is no different.  The authors chose DRAQ5 as the DNA counterstain here and it shows its many capabilities: for the fixed cell assays, for the live cell assays, to confirm cytoplasmic-to-nucleus translocations, to show nuclear morphology (and changes thereof) and nuclear staining intensity (i.e. DNA content as an indicator of altered proliferation) compared to controls.

Patient -derived fibroblasts were chosen due to the accessibility of samples via skin biopsies and also given that fibroblasts have a large cytoplasm.

They chose six assays that reflected changes in important organelles and pathways that would allow tractability to downstream investigations. From these they were able to correlate the VUS of well-known genes with abberancies in the relevant positive controls while in those cases where there was a gene of uncertain significance, differences in one or more of the assay readouts gave direction for onwards investigation. 

What also transpired was that a broader, untargeted approach combining all the ca. 300 features that could be extracted from each assay, including those of the nuclear and brightfield images, proved able to separate samples into defined clusters, despite the susceptibility identified being non-tractable directly from any of the 6 assays - akin to phenotypic screening "hits" in drug discovery, and perhaps specifically the "cell painting" assay. These diagnostic "hits" were then confirmed by orthogonal analysis, for example increased intensity of a nucleus correlating to de novo DNA synthesis in the S phase of the cell cycle and therefore indicating different cell proliferation compared to healthy controls, shown by simple DNA histograms.  These correlated to a common genetic variant.

This work may signal a route to a screening-type strategy as a powerful yet relatively low-cost intermediate gateway to aid the conclusion of a detailed diagnosis with affected pathways that can assist clinicians and patients uncover the landscape of the VUS in question.

Where can I buy DRAQ5?

Reference:

Muffels et al. Imaging flow cytometry‑based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance.  Genome Medicine (2025) 17:12

DOI:10.1186/s13073-025-01433-9

Powerful Spheroid Imaging Methodology using DRAQ5

This new work builds on earlier studies to determine a best methodology to clear and image tumour spheroids (Nürnberg et al. 2020) previously reviewed here.  This showed the performance advantage of a low-cost glycerol-based RI correction and nucleus counterstaining with DRAQ5.  The authors, from the laboratory of Rüdiger Rudolph (Mannheim Univ. of Applied Sciences, DE), described a full workflow from tumor spheroid seeding through treatment, and onto whole mount clearing, staining, immunofluorescence imaging and image analysis.

A significant part of this work was the deployment of a deep-learning-based segmentation tool which could give single cell information within the context of the complete 3D spheroid whole mount rather than the limited information available from a cryosection or a 2D optical section of a spheroid.  One fascinating finding was that it was possible to differentiate the tumor and stromal (fibroblast) cells on the basis of nuclear morphology alone which further simplifies the preparative steps and aids the interrogation of the interactions between two cell types in co-culture.

Pancreatic cell line mono-cultures and co-cultures with fibroblasts were exposed to different cytostatic treatments and show features consistent with previous findings on these drugs and the presence or absence of the co-cultured stromal cells.

The results show strong correlation between the whole mount procedure and cryosections.  Using their procedure, DRAQ5 maintained good penetration and staining of nuclei throughout the spheroids. Where can I buy DRAQ5?

The authors concede a limitation of the study in that these results are on single pancreatic cancer cell line but they signal their intention to test their workflow on patient material with the ultimate goal of informing personalized medicine.

References:
Vitacolonna, Mario, et al. "A spheroid whole mount drug testing pipeline with machine-learning based image analysis identifies cell-type specific differences in drug efficacy on a single-cell level." BMC cancer 24.1 (2024): 1542

Clear sense - optical clearing of microtissues, tissue slices. BioStatus Blog, June 2021.

DRAQ9 segments cytoplasm in LNP localization studies

Led by Prof Efstathios Karathanasis, a group at Case Western Reserve University has developed an improved and tunable technology for the delivery of gene silencing signals (siRNA for example) to achieve PD-L1 gene silencing. The vehicle lipid nanoparticles (LNPs) have modified PEG content that adjusts their cellular uptake.

In one series of experiments to show the difference in uptake based on PEG content mouse dendritic cell line DC2.4 cells were stably-transfected with GFP and adhered to plates. These were then exposed to different formulations of LNPs loaded with GFP-specific siRNA cargo.  After a specified period of exposure to LNPs the cells were stained with Hoechst for the nucleus and DRAQ9™ (1:500 i.e. 2 µM) to determine the GFP signal, inversely proportional to the degree of silencing delivered by the LNPs.

The three fluorescent components used - Hoechst 33342, GFP and DRAQ9™ are ideally suited to a three-colour experiment of this nature allowing trivial spectral separation and cell compartment segmentation.

Where to buy DRAQ9™

Reference:

Lipid Nanoparticles and PEG: Time Frame of Immune Checkpoint Blockade Can Be Controlled by Adjusting the Rate of Cellular Uptake of Nanoparticles.

Andrew S. Choi, Taylor J. Moon, Anubhuti Bhalotia, Aarthi Rajan, Laolu Ogunnaike, Diarmuid W. Hutchinson, Inga Hwang, Aaditya Gokhale, Justin N. Kim, Timothy Ma, and Efstathios Karathanasis.

Molecular Pharmaceutics (2025) Article ASAP

DOI: 10.1021/acs.molpharmaceut.4c01039

RBC manufacture without high EPO costs

The need for a reliable supply of O-negative (universal donor) red blood cells has driven ex vivo RBC manufacture closer to reality in recent years. However, the necessity for erythropoietin (EPO) in the cell culture to promote lineage maturation has remained a significant cost burden. New work from Stanford and UCSF aims to overcome this.

The research utilises protein and genome engineering to create a synthetic EPO receptor (SynEPOR) on HPSCs that is induced by a small molecule rather than by EPO. This is achieved with equivalent performance to EPO but with a 500-fold reagent cost reduction.

In flow cytometric analysis, to determine the differentiation towards fully matured RBCs, DRAQ5™ was employed to show extent of enucleation. Far-red fluorescing DRAQ5™ has been confidently employed in this way for two decades (Fraser, 2005).

The cell-engineering concept described in this work should be transferable to other clinically desirable cells and cell behaviours, to similarly put phenotypic differentiation under the control of small molecule inductions (or suppressions) to achieve a desired terminal cell functionality.

WHERE TO BUY DRAQ5

References:

Shah, A.P., Majeti K.R., et al. "Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis." Nature Communications 16.1 (2025): 1140.

Fraser, S.T., Isern J., and Baron, M.H. "Characterization of Circulating Primitive Erythroid Cells during Embryogenesis Using a Human ε-Globin-GFP Transgenic Mouse Model." Blood 106.11 (2005): 3611.

Exciting New Macrophage Cell Therapy Option

New research published in Nature Communications offers an exciting new cell therapy option using allogeneic macrophage drug conjugates (MDC) to better attack solid tumors, in an effort to overcome some of the limitations experienced by other cell-based therapies.  The work was led by scientists at Cellis AG, a European biotechnology company with focus in cell-based oncology therapy based in Zurich, Warsaw University of Life Sciences and collaborators in Berlin and Edinburgh.

The methodology relies upon a novel mechanism for the preferential in vitro uptake of human heavy chain ferritin (HFt) by human monocyte-derived macrophages (hMDM). This novel clathrin-mediated endocytosis mechanism, in addition to the well-understood transferrin receptor 1 (TfR1/CD71), predominantly utilised a Class A Scavenger Receptor - MSR-1. Both receptors are highly expressed on macrophages. The resulting efficient loading of macrophages was shown not to be cytotoxic to the cells.

Further, central to the therapeutic strategy was the ability to pre-load HFt with small molecule drugs at useful drug/protein ratios. The resulting HFt-drug conjugates were found to be a highly stable resource to load hMDM.

hMDM were found to be the most suitable cell type, importantly of allogeneic origin and which were highly amenable to cryopreservation.

The resulting allogeneic macrophage drug conjugates (MDC) were then tested for their ability to target cancer cells and to determine the mechanism.  MDCs targeted tumor cells and cell killing relied upon Hft-drug conjugate transfer from MDC to tumour cells by direct cell-cell contact by a process termed TRAIN (TRAnsfer of Iron proteiN). A wide range of solid tumour cell lines were tested, with low side-effects, highly targeted to the tumour cells and demonstrating good levels of apoptosis. In a variety of in vivo models, xenograft tumours shrank, cell proliferation was reduced and animals maintained weight.

MDCs were also combined with stand-alone therapeutic agents e.g. checkpoint inhibitors, to significant effect.

DRAQ7™ was used in a variety of assays to determine both unwanted and desired cytotoxicities. DRAQ7™ has ideal properties for development of cell therapies, in cross-platform cell-based assays - time-lapse cell-cell killing assays, product quality assurance analysis and more. It is a chemically-defined reagent manufactured following the ISO 9001:2015 quality system.

WHERE TO BUY DRAQ7

Reference:
Taciak B, Bialasek M, Kubiak M, et al. Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism. Nature Communications ( 2025) 16:1327. https://doi.org/10.1038/s41467-025-56637-9

DRAQ5 for FISH counterstaining intracellular pathogens

In this new work to determine the role of purines in innate immunity a group led by scientists at UCSD used C. elegans orthologs of enzymes in the human purine salvage pathway. 

In one aspect of this work they also treated HUVECs with concentrations of deoxyadenosine with and without anti-IFN antibody and then infected the cells with the intracellular parasite E. intestinalis.

DRAQ5 was used as counterstain for FISH on the HUVEC cells. The analysis contained E. intenstinalis 16S rRNA probe tagged with Quasar 570 chromophore (DsRed channel), Calcofluor White to detect bacterial cell wall (DAPI channel)

DRAQ5 to label nuclei (Cy5 channel).  The data were then used to quantify any significant impact on the infection load in the cells according to the dose of deoxyadenosine, as a measure of enhanced innate immunity.

Due to Calcofluor White's emission being in the DAPI channel DRAQ5 was chosen as DNA counterstain for these FISH experiments, showing its ideal utility for experiments to study intracellular parasitic infections since there is also great value in combining the FISH labeling with Calcafluor White's ability to label the prokaryotic cell wall.

WHERE TO BUY DRAQ5

Reference:
Wernet, N.D., Tecle, E., Sarmiento, M.B., Kuo, C.-J., Chhan, C.B., Baick,
I., Batachari, L.E., Franklin, L., Herneisen, A., Bhabha, G., Ekiert, D.C., Hanna-Rose, W., Troemel, E.R.
Adenosine deaminase and deoxyadenosine regulate intracellular immune response in C. elegans.
ISCIENCE (2025), DOI: https://doi.org/10.1016/j.isci.2025.111950.

Hypotonic vacuoles confirmed by DRAQ9

Fascinating new knowledge on hypotonic vacuoles from a collaborative effort between Kent State Univ, Hamburg and Salzburg.

Despite the well-described and long-understood rationale for vacuoles in lower order eukaryotes little work has been done to determine the conditions for and source of vacuoles in mammalian cell systems. This new work published in Scientific Reports and led by Prof. Michael Model (Kent State Univ.) further classifies watery vacuoles, their origins and includes a newly described vacuole resulting from hypotonic environments and specifically, it transpires, from a low-chloride environment.

To confirm that these indeed were lipid-encapsulated watery vacuoles the cells were labelled with lipophilic probes, including DRAQ9™.

Prof. Model commented in a recent product review: 
“..DiI that we tried before failed to stain the vacuoles, but with DRAQ9 the result was unequivocally positive: a bright staining was easily detectable under a confocal microscope..”

WHERE TO BUY DRAQ9

Reference:
Zook, E., Pan, Y.E., Wipplinger, A. et al. Delayed vacuolation in mammalian cells caused by hypotonicity and ion loss. Sci Rep 14, 29354 (2024).

Myth-busting DNA dyes and single cell sorts (UPDATED)

Sample preparation for single cell and isolated nuclei sorting: de-mystifying the use of DNA dyes for transcriptomics and genomics

Sample preparation is a critical step in the downstream molecular analysis of single cells and nuclei.  This is true for all biological analysis of course, so do the amount of sample preparation your end-point requires and no more!  RNA work especially means much to consider and the purpose of this article is to de-mystify the use of nucleic acid binding dyes in the sorting of target cells and nuclei, a widely used technique to deliver high quality, singlet events onwards for downstream analysis.

What's the problem..?

This article was stimulated by recent community discussion threads that pointed to technical support posts (then repeated and therefore somewhat carelessly validated elsewhere) on single cell sorts for molecular biology that solely recommend 7-AAD as a viability dye to exclude dead cells.  It would appear (strangely) that no others have been tested to support this assertion despite the wide use of other well-known and widely understood and trusted analogous modern reagents in countless peer-reviewed publications.  

Conveniently, this author has many years of experience during the early evolution of modern molecular biology in the 80s and 90s.  That led to a curiosity about the validity of these statements and what the published literature might have to offer in defence or otherwise!  Interestingly, it is, in fact, molecular biology research and some drug discovery that provides the background knowledge.

The take-home message (if you can’t wait) is that you, if you are a shared resource lab or a user, can freely choose from a wide range of DNA binding dyes, be they cell impermeant or permeant.  There are some caveats to this that are explored herein, so do please read on all the same!

What's the evidence..?

Molecular biology research basics of the last 20 years or more give us a very good steer on this topic.  Work from 2000 shows that the classical DNA dye ethidium bromide (Eth-Br) demonstrates interference of Taq polymerase (Taq pol) with an IC₅₀ of 2-200 µM, a level that is similar to other dsDNA mono-intercalators (1).

This concentration was found to be consistent with possible interference of Taq pol by humic acid.  Humic acid is a DNA intercalator, found in soil and consequently often present in scene-of-crime forensic samples (2). This was understandably a critical and obvious concern in the early days of forensic science, asking what in a sample (i.e. assay contaminants) might impact performance of amplification of DNA evidence.

DNA intercalators have a useful role in therapeutics, including cancer e.g. mitoxantrone.  In a search for intercalators as candidate druggable inhibitors of viral reverse transcriptases (RT) similar IC₅₀ were observed.  Meanwhile, in the same work, it was also found that dimers (i.e. so-called bis-intercalators such as ethidium homodimer-1/EtHD-1) potently inhibit RT (at the nanomolar level) especially with limited substrate (3) which one might observe in a poor quality sample.

Some years later the same authors re-confirmed their earlier micromolar IC₅₀ for monomers but were also able to achieve nanomolar RT inhibition with bis-intercalators now designed on very weak monomeric intercalators (4), giving further credence to the general concept of template stabilisation by dimers of intercalators.

In other more tangential work to potentially aid quantitative PCR (QPCR) for enumeration of viable bacteria ethidium & propidium (DNA mono-intercalator) monoazides were used as cell impermeant DNA crosslinkers.  These exhibited stabilising effect on the templates to inhibit PCR of the templates in dead (permeabilised) bacteria.  This was demonstrated at 20 µM, a covalent cross-linking of DNA to exclude those unwanted templates (5), consistent with the IC₅₀ for the early work on bis-intercalator stabilisation of templates.

Exploring further the greater potential impact of bis-intercalators, an Aarhus lab showed clearly that the homo-dimers (examples include TOTO™-3, BOBO™-3, POPO™-3, EtHD-1) and interestingly also the mono-intercalators SyBr® Green and SYTOX™ Orange appear problematic for real-time PCR, likely non-covalently stabilising dsDNA templates.  In their work, the classical methodology of double-stranded template melting temperatures (Cᴛ) were used to measure the stabilising effect of intercalation, with increasing Cᴛ at higher concentrations (ca. 2 µM). Critically, some were considered to be potentially directly toxic to Taq pol, where no harmless concentration could be determined (6).

These publications, therefore, set the background for the potential interference of DNA and RNA template amplification.

What's the reality in a cell or nuclei sort..?

In a suspension of cells or nuclei what is the likely concentration of a DNA intercalator delivered to a RT/PCR reaction in a single cell sort droplet?

From orthogonal methods, we have approximated dsDNA occupancy in isolated nuclei / intact cells for our own mono-intercalators – cell-impermeant viability dye DRAQ7™ and cell-permeant DRAQ5™ respectively.  This approximates to 6 x 10⁶ molecules per cell or nucleus “event” at saturating concentration - a maximal level that is needed for DNA content / cell cycle analysis - circa 4- to 10-fold above the typical concentration required in sorting. This accords to attomoles per nucleated or nucleus event, an insignificant amount in terms of likely contribution to any inhibitory concentration.  

Further, the typical sorting droplet volume (0.8 nl) (A) with dye originally present at 2-20 µM (B) in the sample stream is diluted 1 in 25 (C) by sheath fluid and then massively diluted into the RT reaction well volume of 8 µl (8000 nl) (D).

Thus, the final concentration of DNA intercalating dye:

= B x A/D x 1/C = 2-20µM x 0.8/8000 x 0.04 = 8-80 pM

Overall, here is a 250,000-fold dilution and a final concentration that is a factor of 10⁵-10⁶ below the “inhibitory range” described in the earlier research, and as illustrated in this representative IC₅₀ curve with the inhibitory region shaded in purple and the red arrow showing the relative position of the practical concentration of DNA intercalator in the typical experiment.  Even allowing for batch sorting of events it would need in the region of 10,000 events to begin to reach the inflection of the IC curve.

In our own experience as part of the flow cytometry community we know that this is not a new story for single cell analysis.  In 2004 cytometry pioneer Willem Corver and colleagues sorted tissue digest cells into PCR: DRAQ5 for cell cycle vs. a gene mutation surface marker (7); all done without mishap from a very complex tumour tissue digest milieu.

UPDATE (Aug. 2024): recent experiments at an expert SRL/training centre on the recently-released BD FACSDiscover S8 show that on this instrument the desired concentration of DRAQ5 is 0.5 - 1 µM (for 10⁶ cells/ml), meaning a further significant dose reduction to 2-4 pM. See related article

Subsequently, leading labs in the field who routinely sort cells and nuclei in massive studies (e.g. EMBL, UCSD respectively) utilise a wide range of DNA intercalating dyes, including total/dead combinations such as Hoechst + DRAQ7 (8) or DRAQ5 + DAPI (9) for cell sorting, and DRAQ7 for isolated nuclei (10). The EMBL FACS core facility’s seminal paper (9) begs questions of what can happen to your cell sample – apoptosis, non-lethal stress, mitosis, and so on – that can dramatically impact the transcriptome.  UCSD’s numerous papers, with Sebastian Preissl as the nuclei sorting expert, have standardized robust practice utilizing DRAQ7 as the nuclear event trigger (10) and, prior, combined DRAQ7 with forward scatter to allow sorting of viable cardiac myocytes from a complex cell digest (11).

The take-home message..

Bis-intercalator DNA dyes and the mono-intercalators Sytox™ Orange and SyBr® Green should be avoided in single cell sorting workflows for genomics and transcriptomics based on earlier evidence that is orthogonal to the field of interest here.  

Similarly, one would avoid Live/Dead fixable dyes (unless required for fixed cell workflows) due to limited signal enhancement of positive events over negatives and the risk of possible internal cross-linking.  

The latter is likely implicated with Calcein AM which should be avoided as a positive marker of cell integrity as determined by the altered gene profiles of cells labelled with it (12).  Nonetheless, for off-line evaluation of cell health Calcein AM can be safely used - as combined with DRAQ7 on the BD Biosciences Rhapsody™ platform.

Otherwise, a user should, in principle, be able to use any other mono-intercalator DNA dye for dead cell exclusion, positive single cell event marking (and even cell cycle position sorting) and sorting of isolated nuclei.  Importantly, this allows the user the widest choice of reagents for best fit to the demands of i) sample ii) available platform(s) and iii) sorting panel design components.

Based on this literature review and an investigation of the dose of intercalator delivered to RT or Taq pol reactions in single cell sorting any claims about the requirement for the sole use of 7-AAD as a DNA-binding viability dye would appear to be unfounded.

A Little Reflection..

This blog was precipitated by the assertion of 7-AAD being the only recommended choice of DNA intercalating dye to exclude dead cells for single cell sorting. According to expert opinion, a European BMT reference lab, 7-AAD is now a poor choice for dead cell exclusion.  That lab compared far-red fluorescing viability dye DRAQ7 and 7-AAD in a critical assay - the ISHAGE protocol for CD34 stem cell enumeration - for their ability to clearly define the “snapshot” of three clusters: negatively stained intact cells, an expected intermediate population of momentarily/newly leaky cells and the bright fully-stained dead cells.  The ability of DRAQ7 and inability of 7-AAD in this respect was described in a poster presentation at EBMT in 2013 (13).

One suggestion for the use of 7-AAD might be the cost-saving in using a first- generation viability dye.  The reality however is that the marginal cost saving (likely to be less than one US Dollar) would be dwarfed by costs of any antibodies used and moreover the downstream sequencing procedures and subsequent data analysis.

Most of all, think carefully about the reagents in your workflow.  Could they impact on or interfere with your biology?  Are they compatible with instrumentation options available to you?  Ultimately, do you know what you’re using?  The downstream molecular analysis is very expensive, so avoid shortcuts!

Acknowledgements 

Specialist technical knowledge was kindly provided by Christopher Hall MSc, Babraham Institute and Paul J Smith Prof. Em., Cardiff University.

References

1. Nath, K., Sarosy, J. W., Hahn, J., & Di Como, C. J. (2000). Effects of ethidium bromide and SYBR® Green I on different polymerase chain reaction systems. Journal of biochemical and biophysical methods, 42(1-2), 15-29.

2. Thompson, R. E., Duncan, G., & McCord, B. R. (2014). An investigation of PCR inhibition using Plexor®‐Based quantitative PCR and short tandem repeat amplification. Journal of forensic sciences, 59(6), 1517-1529.

3. Jain, N., Francis, S., & Friedman, S. H. (2012). Inhibition of therapeutically important polymerases with high affinity bis-intercalators. Bioorganic & medicinal chemistry letters, 22(14), 4844-4848.

4. Jain, N., & Friedman, S. H. (2019). Multiple weak intercalation as a strategy for the inhibition of polymerases. Bioorganic & medicinal chemistry letters, 29(3), 424-429.

5. Krüger, N. J., Buhler, C., Iwobi, A. N., Huber, I., Ellerbroek, L., Appel, B., & Stingl, K. (2014). “Limits of control”–crucial parameters for a reliable quantification of viable campylobacter by real-time PCR. PloS one, 9(2), e88108.

6. Gudnason, H., Dufva, M., Bang, D. D., & Wolff, A. (2007). Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Research, 35(19), e127.

7. Douwes Dekker, P. B., Corver, W. E., Hogendoorn, P. C., van der Mey, A. G., & Cornelisse, C. J. (2004). Multiparameter DNA flow‐sorting demonstrates diploidy and SDHD wild‐type gene retention in the sustentacular cell compartment of head and neck paragangliomas: chief cells are the only neoplastic component. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland, 202(4), 456-462.

8. Klingler, E., De la Rossa, A., Fièvre, S., Devaraju, K., Abe, P., & Jabaudon, D. (2019). A translaminar genetic logic for the circuit identity of intracortically projecting neurons. Current Biology, 29(2), 332-339.

9.     Ordoñez‐Rueda, D., Baying, B., Pavlinic, D., Alessandri, L., Yeboah, Y., Landry, J. J., ... & Paulsen, M. (2020). Apoptotic Cell Exclusion and Bias‐Free Single‐Cell Selection Are Important Quality Control Requirements for Successful Single‐Cell Sequencing Applications. Cytometry Part A, 97(2), 156-167.

10. Preissl, S., Schwaderer, M., Raulf, A., Hesse, M., Grüning, B. A., Köbele, C., ... & Gilsbach, R. (2015). Deciphering the epigenetic code of cardiac myocyte transcription. Circulation research, 117(5), 413-423. (See suppl. data).

11. Zhang, K., Hocker, J. D., Miller, M., Hou, X., Chiou, J., Poirion, O. B., ... & Ren, B. (2021). A single-cell atlas of chromatin accessibility in the human genome. Cell, 184(24), 5985-6001.

12. De Micheli, A. J., Laurilliard, E. J., Heinke, C. L., Ravichandran, H., Fraczek, P., Soueid-Baumgarten, S., ... & Cosgrove, B. D. (2020). Single-cell analysis of the muscle stem cell hierarchy identifies heterotypic communication signals involved in skeletal muscle regeneration. Cell reports, 30(10), 3583-3595.

13. Moshaver, B., Huys, E., Terwindt, E., Kramer, P. A., & Preijers, F. (2013, April). DRAQ7, a novel viability dye to determine the correct amount of existing dead and apoptotic cells in CD34+ stem cell enumeration. In Bone Marrow Transplantation (Vol. 48, pp. S182-S183). London, England: Nature Publishing Group.

Roy Edward, FRMS       

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