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