Monday, 20 October 2025

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. Science389(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

Monday, 6 October 2025

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.


Friday, 3 October 2025

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.