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.

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Reference:

Yosief, Robel, Giammarco Nebbioso, Vasilij Koshkin, Yumin Qiu, Chun Peng, Vadim Elisseev, and Sergey Krylov. "Making Cytometry of Reaction Rate Constant (CRRC) Applicable to Motile Cells." (2022). Preprint on ChemRxiv, 21 April 2022. DOI:10.26434/chemrxiv-2022-gtgzf

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.

Single tube 3-parameter apoptosis assay

A simple, yet improved (3-parameter, 3-colour) apoptosis assay 

The combination of Annexin V binding to detect phosphatidyl serine displayed on inverted plasma membranes of early apoptotic cells and a DNA-binding viability dye that permeates leaky plasma membranes denoting very late apoptotic/dead cells is a widely used simple, low-cost, single tube assay for cellular apoptosis.  

However, it is clear that these two plasma membrane "events" give little information about the actual events and progress of apoptosis and it would be therefore beneficial to include a further parameter that is earlier, independent, sentinel or pivotal for apoptosis or more correctly, programmed cell death.  

One such parameter might be the collapse of mitochondrial membrane potential (ΔΨm) observed proportionally with the reduction of binding of a commonly used rhodamine-based dye: TMRE (or TMRM).  

Historically, the most commonly used combination of Annexin V and viability dye has been Annexin-V-FITC and propidium iodide.  Unfortunately, the emission spectra of propidium iodide and TMRE/TMRM heavily overlap making this combination impractical.  

However, one can simply replace propidium iodide with the far-red fluorescing DNA-binding viability dye DRAQ7, which then permits combination of viability dye with TMRE and Annexin V enables the three parameters to be measured in a single-tube experiment.

An example of this, shown below, has been kindly provided by Dr. Lucia Piñon P Giraldez, Head of Advanced Light Imaging and Flow Cytometry Facilities, MRC Toxicology Unit, Cambridge, UK.  Annexin V is detected in the "FITC" channel, TMRE in "PE" and DRAQ7 in "APC-Cy7".

One might also consider caspase 3/7 activation reporters. These have, similarly, been combined with TMRM and DRAQ7 and demonstrated in time-lapse image-based assays.

DRAQ7 offers new assay design alongside consistent product quality and ease-of-use backed by 1700+ citations (as of Nov. '23) in peer-reviewed journal articles.

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DRAQ7™ offers a significant advantage in that it is truly cross-platform compatible, able to be the cell death reporter in flow cytometry, plate-based cytometers (Sartorius CeligoS), imaging flow cytometry (e.g. Cytek® Amnis® Imagestream®, real-time imaging analysis (e.g. on the Sartorius Incucyte®), a high content imaging platform (e.g. Revvity Opera Phenix®, Molecular Devices ImageXpress®), organ-on-a-chip systems, or the various fluorescence microscopes, with or without live cell capabilities, depending on your application’s need for time-lapse or simple end-point measurements. Importantly, this means you can switch an assay between platforms according to your research’s changing needs with limited disturbance in the reagents used.

References:

Wlodkowic, D., .. & Darzynkiewicz, Z. (2013). Kinetic viability assays using DRAQ7 probe. Current protocols in cytometry, 65(1), 9-41.

Deo, P., .. & Lithgow, T. (2018). Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis. PLoS pathogens, 14(3), e1006945.

Automated, Multi-Parameter, Kinetic Methods to Quantify Cell Death - Application Note. Brad Larson, Principal Scientist, BioTek Instruments, Inc., Winooski, VT USA. March, 2018

Jost, T., .. & Hecht, M. (2022). Influence of alectinib and crizotinib on ionizing radiation-in vitro analysis of ALK/ROS1-wildtype lung tissue cells. Neoplasia, 27, 100780.