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

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

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

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       

© Copyright BioStatus Limited 2024

SYTOX, TOTO, BOBO, POPO and SyBr are trademarks of Thermo Fisher Scientific, Inc. Rhapsody is a trademark of Becton, Dickinson and Company

DRAQ5 and FACSDiscover S8 (updated)

BioStatus been working with the BD FACSDiscover S8 at a leading SRL and training site on the use of DRAQ5™, as described in the article that described this novel instrument's capabilities.

In the course of this work it was quickly determined that the production model, not surprisingly, significantly outperforms the experimental platform utilised in that original Science journal article (1). 

Central to this, sensitivity is markedly different.  We determined that the optimal concentration for DRAQ5 is 0.5 - 1.0 µM for a suspension of 1x10⁶ cells/ml.

Therefore, DRAQ5 will be the preferred choice in most applications since at this concentration it is highly economical and delivers desirable and well-defined nuclear labeling in the far-red.  This fluorescence property releases the two other fluorescence channels for further biological parameters.

DRAQ5 is already validated in many hundreds of imaging flow cytometry papers using the Imagestream X mark II (CyTEK Biosciences) (2) and its common use in preparative single cell /nuclei sorts for transcriptomic and genomic analyses (3).  These are two techniques that, for example, the BD FACSDiscover S8 is able to combine.

Importantly, all users of DRAQ5 can be assured of its quality and consistency maintained over many years of experience at BioStatus, and the global availability of the product through carefully selected channel partners or, as always, direct from BioStatus. (Buy DRAQ5 here)

NOTE: We recommend the BioStatus product SKU: DR05500 (500 µl; 500 µM) for convenient pipetting of between 1 and 2 µl directly into each ml of cell suspension (to achieve a 0.5 -1.0 µM final concentration, as above).  This product is ideally formulated for use on the BD FACSDiscover S8 and is available direct from BioStatus.

It should be borne in mind that the concentration maybe somewhat different for different cell types but it is unlikely to vary by more than 2-3 fold, given the experience of the 12,000+ citations for DRAQ5 in the published literature, across a very broad array of human cells.

These concentrations fall even further below those already determined to be entirely compatible with downstream RT-PCR and PCR amplifications, as described here.

Also, it should be possible to use the related DNA dye CyTRAK Orange™, with the caveat that this has peak emission at 610 nm with a differential staining of nucleus and cytoplasm (shown to be segmentable by intensity masking and a watershed, respectively, in microscopy).  We have not tested CyTRAK Orange yet on the FACSDiscover S8 but would predict that it might require a concentration of 1 - 2 µM, based on experience of a wide variety of cell analysis platforms and from what we have already learned about DRAQ5 on the FACSDiscover S8.

This story will be expanded on in the coming months.

References:

1. Schraivogel D, Kuhn TM, Rauscher B, Rodríguez-Martínez M, Paulsen M, Owsley K, Middlebrook A, Tischer C, Ramasz B, Ordoñez-Rueda D, Dees M. High-speed fluorescence image–enabled cell sorting. Science. 2022 Jan 21;375(6578):315-20.

2. Harte DS, Lynch AM, Verma J, Rees P, Filby A, Wills JW, Johnson GE. A multi-biomarker micronucleus assay using imaging flow cytometry. Archives of Toxicology. 2024 Jul 12:1-7.

3. Ordoñez‐Rueda D, Baying B, Pavlinic D, Alessandri L, Yeboah Y, Landry JJ, Calogero R, Benes V, Paulsen M. Apoptotic Cell Exclusion and Bias‐Free Single‐Cell Selection Are Important Quality Control Requirements for Successful Single‐Cell Sequencing Applications. Cytometry Part A. 2020 Feb;97(2):156-67.

BD FACSDiscover S8 and BD are trademarks of Becton, Dickinson and Company

© Copyright BioStatus Limited 2024

Organ on a chip and DRAQs

Organ-on-a-chip (OOAC) technologies and DRAQs

DRAQ5™, DRAQfx™, DRAQ7™, DRAQ9™ and HypoxiTRAK™ offer a number of special benefits for the analysis of the improved physiological relevance organ-on-a-chip technologies (OOAC) or microphysiological systems (MPS).  The red excitation and the far-red fluorescence reduces the interference from sample autofluorescence and the improved excitation light penetration and signal fluorescence transmission, as a direct consequence of the longer wavelengths involved.

DRAQ7™, the far-red DNA-binding viability dye, has been widely demonstrated for the time-lapse reporting of cell death in a wide variety of OOAC platforms, of 2D and 3D cell geometries.  This is consistent with its cross-platform capabilities demonstrated on flow cytometers, imaging flow cytometers, fluorescence microscopy and high content screening platforms. DRAQ7™ breaks the problem of the use of homogeneous assays such as Alamar Blue, ATP, MTT to measure cell viability which all necessitate extra destructive processing of samples and cannot be applied in real-time / time-lapse.

DRAQ5™, the widely-used far-red, live-cell permeant DNA counterstain, is backed by 12,000+ publications across a wide range of applications.  Its major use is as an endpoint live-cell or fixed-cell counterstain and in addition to slide-based immunofluorescence microscopy has been widely utilised in OOAC imaging workflows.

Given the variety of platforms and scaffolds that are being employed it is important to have alternative chemistries for fixed-cell endpoint analyses.  Accordingly, using the same fluorescence Ex/Em settings one can choose between hydrophobic DRAQ5™ or hydrophilic DRAQfx™. This allows for easier switching between platform technologies, without having to re-design the analysis set-up.  

Segmentation of 3D objects can be challenging and to address this BioStatus has developed a non-toxic, log-term probe – DRAQ9™ - that can be used to define the cellularity of a 3D microtissue/spheroid/organoid and track its changes over many days, to enable automatic masking without the confounding aberrations of ECM protrusions and to follow cell migration.

A related fluorescent probe HypoxiTRAK™ is a novel means to report the response and accumulated experience of individual cells to hypoxia.  This far-red probe is otherwise non-toxic to cells and is only bioconverted to accumulate inside a cell when that cell experiences the paucity of dioxygen (below 3%; the probe’s direct competitor).  HypoxiTRAK is able to report on hypoxia experience over many days in 2D culture and 3D microtissues/organoids. 

You can purchase the products direct from BioStatus (and take advantage of our no-quibble, money-back guarantee) or from our carefully-selected channel partners. 

A selection of citations follows by product with disease/tissue and lead institution:

DRAQ5™ (most recent papers only)

Pamies et al. Altex 2017        Brain MPS         Johns Hopkins Univ.

Scheinpflug et al. Lab Chip 2023         Bone scaffolds   Greifswald Univ.

Vanderlaan et al. Lab on a Chip 2023  Islet-on-a-chip Purdue Univ.

Signore et al. Sens Biosens Res 2021  Gut-on-a-chip CNR-IMM, Lecce

Huebsch et al. Nat Biomed Eng 2022  Cardiac chip UC-Berkeley

Arakawa et al. Comms Biol 2023 Bile canaliculus   Kanazawa Univ.

Hansen et al. Circ. Res. 2010 Eng. heart tissue     Hamburg-Eppendorf Univ.

Zhang et al. Lab Chip 2022 Dental stem cells Hong Kong Univ.

Järvinen et al. Adv Funct Mater 2020 Hepatocytes (2/3D) Univ. Helsinki

Pieters et al. Biofabrication 2022 Adipose (3D) Univ. Toronto

Afshar et al. Sci Rep 2020 Skeletal muscle   Univ. Toronto

Orellano et al. Adv Funct Mater 2023 Cell bioprinting   Charité, Berlin

Amiri et al. Adv Sci 2021 Blood-Brain Barrier UC-Berkeley

DRAQ7™

Tran et al.  Transplantation 2023 Organ rejection         Univ Hosp Geneva

Geyer et al. Front Immunol 2023 PDAC          Mimetas

Law et al. Adv Sci 2022 Breast cancer         Univ Tech Sydney

Miller et al. Tibtech 2020 Intestine         JHU / U-WA

Boquet-Pujadas et al. Sci Adv 2022 Intestine         Univ Paris Cité

Kopec et al. J Toxicol Sci 2021 Liver         Pfizer

Bray et al. Front Bioeng Biotech 2019 Liver tumors         QUT, Brisbane

Xue et al. Cell 2023 Tumorspheres         Cypre, Inc.

Willi et al. Front Bioeng Biotech 2022 Ex vivo intestinal       FBRI, Roanoke

Miller et al. Neoplasia 2023 RCC on a chip         U-WA

DRAQ9™

Edward et al. SLAS Conf. 2020 Tumorspheres         BioStatus/Cardiff U.

HypoxiTRAK™

Close & Johnston. SLAS Disc. 2022 HNC spheroids         Pittsburgh Univ.

#OOACT #OOACN #MPS

DRAQ7 assay replaces MTT

As has been found with 7-AAD in the ISHAGE assay, there are more up-to-date and designed-for-purpose reagents available that offer better options for getting the answers needed.  Here, it is the turn of an old workhorse assay using MTT to be under the spotlight of academic scrutiny. Again, the far-red viability dye DRAQ7™ outperforms and simplifies.

Polyphenols have attracted attention as possible anti-cancer agents.  This is particularly true for the gliomas and given the limited success of improving survival rates, such naturally occurring compounds have added potential.

It is common to test compounds for cytotoxic effects using the so-called “Gold” standard homogeneous plate-based MTT assay, an indirect measure of cell viability.  This assay relies upon the bio-conversion of MTT to a purple, insoluble and crystalline formazan product by the remaining healthy, metabolically-competent cells. The assay is stopped by removal of substrate and resolving the crystal deposits with DMSO and the total signal for each well recorded.  The viability of test samples is then referenced against the readings for the negative and positive controls. 

However, the MTT assay is confounded by i) the intensely coloured polyphenols, which overlap the spectra for the formazan ii) the observation of a hormesis effect on glioma cells at high concentrations of certain polyphenols and iii) the potential for DMSO toxicity.

To overcome this, a team from London led by the Francis Crick Institute, turned to flow cytometry as an alternative approach, using the DNA binding viability dye DRAQ7™ to enumerate the live (DRAQ7-negative) and dead (DRAQ7-positive events) for each treatment and relevant controls.  This assay has the advantages that i) DRAQ7™ completely avoids the spectral overlap of polyphenols experienced with the MTT assay, ii) it is a direct measure of cell viability, cell-by-cell, since DRAQ7™ accumulates in the nucleus of dead/dying cells due to associated failure of the plasma membrane and iii) it offers a simplified workflow since DRAQ7™ enables a no-wash procedure.

To underpin the study, a manual microscopic count for cell viability was also performed for a range of concentrations of the different polyphenols and cell lines tested.

Only with the DRAQ7™-based flow cytometry assay were the authors able to get reliable and robust IC curves and IC₅₀ values, and that accorded with the manual microscopic counts.

Because DRAQ7™ is a cross-platform compatible reagent such an assay could equally be performed with fluorescence microscopy, on a high content imaging instrument for example.  This would enable the DRAQ7™ approach to be optimally amenable to adherent or non-adherent cell types as required.  In either case, the viability reporting could be additionally multiplexed with another cellular readout pertinent to the agents being tested, for example mitochondrial membrane potential and/or Annexin V binding as further measures of apoptotic processes, morphometric changes to the cells (forward/side scatter or BF features) or indeed to the desired therapeutic "event" such as a protein translocation (in the case of an image-based assay).   This is economically important since such multiplexing allows a massive reduction in the cost of reagent (notably the cells and their culture).

DRAQ7™ is already widely used in the described manner to report on desired (and undesired) toxicity in a wide variety of assays.  Evident from this work, it would require little optimisation for it to be adopted as the replacement of the indirect MTT assay across a swathe of viability assays, for adherent or suspension cells and only necessitating access to a simple flow cytometer (preferably with plate-loading and well sampling), an automated fluorescence microscopy (high content imager) or a plate-based cytometer (such as the Sartorius Celigo S) and capability of red-excitation (e.g. 594, 635, 647 nm) and deep-red fluorescence detection (e.g. Cy5, Cy5.5, 675LP, 730/50 or similar) which are all commonly found in the drug discovery setting.

WHERE TO BUY DRAQ7

Reference:

Rooprai et al. Anticancer research 40: 5427-5436 (2020) 

 

ASSCR 2023 - Spotlight on Cell Therapies

BioStatus products DRAQ5™ & DRAQ7™ are, of course, used in countless research articles in pursuit of novel cell therapies.  They are trusted by pharma, biotechs and leading research institutes globally.

All BioStatus products are manufactured to the highest standards, from synthesis to packaging, under ISO 9001:2015 certificated processes.

But why should YOU choose them for development, characterisation and quality assurance of cell therapies?

        Where to buy: Sapphire Bioscience - please visit their booth!

To help you, here are some key examples where they have been components of flow cytometric analysis and cell sorting relevant to cell therapies, with hyperlinks to detailed documents and citations:

• Phenotypic analysis: easily gate singlet nucleated cells from doublets and debris, &..
• Cell Cycle / DNA Content Analysis: no-lyse, no wash direct method with minimal sample prep

• Side population analysis: exclude dead cells from disturbing the data

• Monocyte purity check: in preparation of therapeutic macrophages 

• Determining very rare antigen-specific T-cells

• ISHAGE CD34+ Stem Cell Enumeration: more reliable gating 
• cell therapy QC review article
• poster from European ref. lab.

• DNA Double-Strand Break Detection: assessing DSB repair capacity post-cryopreservation (IFC) 

• Micronucleus Detection: high-throughput genotoxicity testing (IFC)

• Smarter Cell Health and Apoptosis Assays: allowing multi-parameter and temporal options

• Single Cell Analysis: industry-standard gating of cells or nuclei for transcriptomics & genomics

• Cell-killing capacity: temporal monitoring of cytotoxic function

• Phagocytosis function: track fluorescence from prey to predator cells

DRAQ7™ to the rescue..

Did you know? You can add DRAQ7™ to a pre-existing chromophore panel when it is later realised a viability dye is needed!  Or, you can use the same concept to create a virtual channel that simplifies any compensation challenges for the other components or even to simply extend the capacity of your cytometer!

See how here

Fluorescence microscopy and high-content imaging

DRAQ5™ has been cited in many thousands of articles as the nuclear counterstain for fixed and live end-point fluorescence microscopy and in high-content imaging screens for drug discovery. 

DRAQ7™ enables time-lapse monitoring of cell health, for many days without inherent toxicity yet faithfully reporting cell death events.

ASK US! If you have any questions or need to discuss a specific assay we are only too happy to help out. Send an email to: enquiry@biostatus.com

        Where to buy: Sapphire Bioscience - please visit their booth!

IFC = Imaging Flow Cytometry

#ASSCR2023

Autophagy UK Network Meeting 2022

BioStatus is delighted to once more support the Autophagy UK Network's annual meeting at the University of Warwick

Here are some dedicated resources for cell biology assays in the study of autophagy, mitophagy, ..

First, a few numbers: you can use BioStatus products with confidence, being cited in more than 12,000 peer-reviewed articles.  DRAQ5 and DRAQ7 have been cited in more than 1200 articles mentioning autophagy.

BioStatus's patented products are all UK inventions, manufactured in the UK, available direct for next-day delivery but also through carefully selected distribution partners.

Here are quick Google Scholar searches to help you.. 

Recent articles with autophagy in the title and using DRAQ5

Recent articles with autophagy in the title and using DRAQ7

DRAQ5 - the far-red, live-cell permeant DNA dye - enables multi-colour flow cytometry, fluorescence microscopy and high content imaging.  At its simplest it's a counterstain for nucleated object gating or identification while at its most sophisticated it's a live-cell reporter of cell cycle.  If you need cryo-EM then it is the perfect fiduciary marker that can bridge living tissue to ultrastructure (see blog).

DRAQ7 - the far-red, viability dye - allows you to add a viability dye into a flow cytometry panel where there doesn't seem to be room for one (see CYTO 2018 poster) or just enables smarter single tube apoptosis assays like AnnexinV / TMRM / DRAQ7 (see blog).  It's become a reagent of choice in time-lapse imaging of cell death / survival in 2D and 3D cultures, being non-toxic and non-interfering (see BLSW 2015 poster and as used in the lab of the opening keynote speaker Prof Sylvie Urbé [Liang, et al. 2015]).

What else can BioStatus offer your research?

Single cell genomics & transcriptomics..

Both DRAQ5 & DRAQ7 feature strongly in single-cell and single-nuclei sorting protocols to aid delivery of quality templates for amplification and sequencing (see blog).

Hypoxia & autophagy..

HypoxiTRAK enables the time-lapse reporting of the accumulated hypoxic experience of individual cells in both 2D and 3D cultures, in hypoxia chambers and by imaging or flow cytometry.  HypoxiTRAK's signal is consistent with Hif-1a expression.  HypoxiTRAK requires no antibody detection nor does it futile redox cycle unlike the nitroimadazole based chemistries. (see blog).

High resolution imaging..

If you need to immobilize cells, model organisms or microtissues to image these at high resolution for colocalizations, FRAP or organelle detail then CyGEL may provide the answer.  This thermo-reversible hydrogel is compatible with live cells and tissues and has ideal optical properties - clear, RI-matched to biological samples, negligible fluorescence.  

Live cell tracking..

DRAQ9 - a novel non-toxic, far-red cell probe to follow cell migration in time-lapse studies (see MMC 2019 poster)

Any questions?  Email us: enquiry@biostatus.com or visit www.biostatus.com

CRRC for motile cells

Scientists at York University, Toronto, led by Prof. Sergey Krylov have developed a methodology that permits the tracking of motile cells for the measurement of Cytometry of Reaction Rate Constant (CRRC). 

In essence, CRRC is a valuable tool in understanding tumour cell biology.  One can measure the difference in a chemical reaction's rate between individual cells in a tumour population.  This can act as a parameter to aid description of the relative proportions of bulk tumour cells and tumour-initiating cells for their reaction kinetics in resistance to chemotherapy, for instance, that might be modelled by clearing or metabolising a drug-like small molecule as the reporter.  Typically, this reporter is fluorescent e.g. fluorescein.

Amongst the challenges that arise are how to robustly segment cells to be measured in a time-lapse fashion, when fluorescence alone might at some point not be sufficiently detectable to describe the cell boundary and, perhaps more significantly, when cells are motile and the "masked" boundary no longer accords with the cell at a later time point.  

To address these concerns the authors (Yosief et al.) tested different transmitted light methods to determine the cell boundaries and settled upon brightfield (BF) as the one of choice.  However, this brought a further potential issue due to the difference in the focal planes of BF and fluorescence (ca. 10 µm) that might impact on the fluorescence quantitation of increasingly out of focus emitted light.  Helpfully, however, this difference accords closely with a typical cell diameter and they embarked on tests to calculate the signal loss at distances away from coincident focal planes.  As a label of fluorescence, cells were incubated with the far-red fluorescent cytoplasmic probe DRAQ9™ and the emitted signal captured for the coincident and non-coincident (+/-) focal planes of fluorescence and transmitted light (BF).  It transpired that the 10 µm offset had a minor impact on the quantitation of fluorescence.

DRAQ9™ staining of cells was simple.  DRAQ9™ was applied to the cells at a final concentration of 2 µM for 30 minutes before excess was removed by washing (3x) and then imaged using epifluorescence microscopy.  DRAQ9™ was detected using a Cy5 cube.

WHERE TO BUY

Reference:

Nebbioso G, Yosief R, Koshkin V, Qiu Y, Peng C, Elisseev V, Krylov SN. Automated identification and tracking of cells in Cytometry of Reaction Rate Constant (CRRC). Plos one. 2023 Jul 3;18(7):e0282990.

Future prospects: 

DRAQ9™ has been shown to be non-toxic over many days exposure to cells at 2 µM both for tracking growth of spheroid microtissues and in scratch-wound motility assays (manuscript in preparation).  Thus, this may present the opportunity to combine it with spectrally-compatible, fluorescently-tagged reporting targets of aggressive tumour cells' metabolism e.g. fluorescein (used in this work), FITC-conjugates, naturally fluorescing pharmacophores (topotecan, hoechst, etc.) as indicators of multi-drug-resistance, and so on.  Indeed, in theory, it should be possible to multiplex reporters for cellular response to combination chemotherapy due to the spectral space afforded by DRAQ9's far-red fluorescence.  This use of DRAQ9™ to demark the cell boundary with fluorescence rather than BF would further reduce any impact of focal plane offset since all measurements - reporter(s) and cell boundary - would be of fluorescence emissions.  This may become more important in cell models where artefacts due to the production of ECM are confounding to automated cell segmentation using BF.