Accurate quantification of apoptosis progression and toxicity using a dielectrophoretic approach

A loss of ability of cells to undergo apoptosis (programmed cell death, whereby the cell ceases to function and destroys itself) is commonly associated with cancer, and many anti-cancer interventions aim to restart the process. Consequently, the accurate quantification of apoptosis is essential in understanding the function and performance of new anti-cancer drugs. Dielectrophoresis has previously been demonstrated to detect apoptosis more rapidly than other methods, and is low-cost, label-free and rapid, but has previously been unable to accurately quantify cells through the apoptotic process because cells in late apoptosis disintegrate, making cell tracking impossible. In this paper we use a novel method based on light absorbance and multi-population tracking to quantify the progress of apoptosis, benchmarking against conventional assays including MTT, trypan blue and Annexin-V. Analyses are performed on suspension and adherent cells, and using two apoptosis-inducing agents. IC50 measurements compared favourably to MTT and were superior to trypan blue, whilst also detecting apoptotic progression faster than Annexin-V

Casein Kinase 1 Underlies Temperature Compensation of Circadian Rhythms in Human Red Blood Cells

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles

Optimisation of dielectrophoretic based molecular targeting for blood chronobiology.

Dielectrophoresis (DEP) is an electrostatic technique which can be used to examine cellular electrophysiology. This method offers many advantages in characterising a cell population over conventional methods; however, it has yet to see mainstream pharmacological application. This work demonstrates a DEP assay, 3DEP, as a viable method in the electrophysiological measurement of cells, its application for measuring drug effects, and more specifically, its use as a molecular targeting tool in circadian research. The reliability of the assay was investigated measuring DEP response of several cell types with high resolution spectra and the operational limits of automatic fitting algorithms were determined for single and multiple populations. It was found that reliable readouts from 3DEP could be achieved in as little as 6 s. The ability of 3DEP to test drug cytotoxicity on Jurkat cells was tested with doxorubicin. DEP measured IC50 values compared well with previoiusly measured colorimetric assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) experimental results, suggesting 3DEP could provide an alternative way to measure molecular events due to drug intervention. With 3DEP having been appropriately optimised, it was then applied to the study of RBC circadian rhythm through both human and vole in vitro and in vivo studies. Disruption of these rhythms can lead to negative health outcomes related to areas such as metabolism, cardiovascular health, and mental health. Since RBCs are anucleated, the mechanism cannot rely on the typically accepted transcription-translation feedback loop found for other cells. 3DEP detected robust rhythmicity in the electrophysiological properties of RBCs in isolated and whole blood samples. These rhythms were altered when introduced to pharmacological intervention. Taken together, these findings suggest a model for the non-transcriptional clock in RBCs. Many applications of the 3DEP system were investigated with respect to molecular targeting highlighting 3DEP’s utility and limitations as a biological tool in research

How can you demonstrate and assess the impact of your publications?

For the researcher who is committed to producing impactful research, who has carefully considered all the advice and has worked hard to put the best strategies into practice, there is another piece of the puzzle to be considered. What evidence can we find that all of our hard work has had an effect? Without that evidence, assessing how successful we have been in meeting our personal objectives presents a challenge. Just as importantly, we may struggle to demonstrate the impact of our research to those that fund us, directly or indirectly – our universities, our funding bodies, and our governments.In this chapter you will:Be introduced to the key ‘worlds’ of bibliographic data and the different types of metric used in research assessmentContemplate the strengths and limitations of different metrics from the perspective of the individual researcherConsider the importance of deploying metrics responsiblySee how metrics can be used to establish specific and measurable objectives for the impact of your publicationsBe encouraged to continually assess and evolve your publication strategy using the insights metrics can provide</ul

Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP)

Contactless dielectrophoresis (cDEP) is a recently developed method of cell manipulation in which the electrodes are physically isolated from the sample. Here we present two microfluidic devices capable of selectively isolating live human leukemia cells from dead cells utilizing their electrical signatures. The effect of different voltages and frequencies on the gradient of the electric field and device performance was investigated numerically and validated experimentally. With these prototype devices we were able to achieve greater than 95% removal efficiency at 0.2–0.5 mm s−1 with 100% selectivity between live and dead cells. In conjunction with enrichment, cDEP could be integrated with other technologies to yield fully automated lab-on-a-chip systems capable of sensing, sorting, and identifying rare cells

Optical measurements of vocal fold tensile properties: Implications for phonatory mechanics

In voice research, in vitro tensile stretch experiments of vocal fold tissues are commonly employed to determine the tissue biomechanical properties. In the standard stretch-release protocol, tissue deformation is computed from displacements applied to sutures inserted through the thyroid and arytenoid cartilages, with the cartilages assumed to be rigid. Here, a non-contact optical method was employed to determine the actual tissue deformation of vocal fold lamina propria specimens from three excised human larynges in uniaxial tensile tests. Specimen deformation was found to consist not only of deformation of the tissue itself, but also deformation of the cartilages, as well as suture alignment and tightening. Stress–stretch curves of a representative load cycle were characterized by an incompressible Ogden model. The initial longitudinal elastic modulus was found to be considerably higher if determined based on optical displacement measurements than typical values reported in the literature. The present findings could change the understanding of the mechanics underlying vocal fold vibration. Given the high longitudinal elastic modulus the lamina propria appeared to demonstrate a substantial level of anisotropy. Consequently, transverse shear could play a significant role in vocal fold vibration, and fundamental frequencies of phonation should be predicted by beam theories accounting for such effects

Contactless dielectrophoretic spectroscopy: Examination of the dielectric properties of cells found in blood

The use of non-invasive methods to detect and enrich circulating tumor cells (CTCs) independent of their genotype is critical for early diagnostic and treatment purposes. The key to using CTCs as predictive clinical biomarkers is their separation and enrichment. This work presents the use of a contactless dielectrophoresis (cDEP) device to investigate the frequency response of cells and calculate their area-specific membrane capacitance. This is the first demonstration of a cDEP device which is capable of operating between 10 and 100 kHz. Positive and negative dielectrophoretic responses were observed in red blood cells, macrophages, breast cancer, and leukemia cells. The area-specific membrane capacitances of MDA-MB231, THP-1 and PC1 cells were determined to be 0.01518 ± 0.0013, 0.01719 ± 0.0020, 0.01275 ± 0.0018 (F/m2), respectively. By first establishing the dielectrophoretic responses of cancerous cells within this cDEP device, conditions to detect and enrich tumor cells from mixtures with non-transformed cells can be determined providing further information to develop methods to isolate these rare cells

Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP)

Contactless dielectrophoresis (cDEP) is a recently developed method of cell manipulation in which the electrodes are physically isolated from the sample. Here we present two microfluidic devices capable of selectively isolating live human leukemia cells from dead cells utilizing their electrical signatures. The effect of different voltages and frequencies on the gradient of the electric field and device performance was investigated numerically and validated experimentally. With these prototype devices we were able to achieve greater than 95% removal efficiency at 0.2–0.5 mm s−1 with 100% selectivity between live and dead cells. In conjunction with enrichment, cDEP could be integrated with other technologies to yield fully automated lab-on-a-chip systems capable of sensing, sorting, and identifying rare cells

Optical measurements of vocal fold tensile properties: Implications for phonatory mechanics

[[abstract]]n voice research, in vitro tensile stretch experiments of vocal fold tissues are commonly employed to determine the tissue biomechanical properties. In the standard stretch-release protocol, tissue deformation is computed from displacements applied to sutures inserted through the thyroid and arytenoid cartilages, with the cartilages assumed to be rigid. Here, a non-contact optical method was employed to determine the actual tissue deformation of vocal fold lamina propria specimens from three excised human larynges in uniaxial tensile tests. Specimen deformation was found to consist not only of deformation of the tissue itself, but also deformation of the cartilages, as well as suture alignment and tightening. Stress–stretch curves of a representative load cycle were characterized by an incompressible Ogden model. The initial longitudinal elastic modulus was found to be considerably higher if determined based on optical displacement measurements than typical values reported in the literature. The present findings could change the understanding of the mechanics underlying vocal fold vibration. Given the high longitudinal elastic modulus the lamina propria appeared to demonstrate a substantial level of anisotropy. Consequently, transverse shear could play a significant role in vocal fold vibration, and fundamental frequencies of phonation should be predicted by beam theories accounting for such effects

Contactless dielectrophoretic spectroscopy: Examination of the dielectric properties of cells found in blood

The use of non-invasive methods to detect and enrich circulating tumor cells (CTCs) independent of their genotype is critical for early diagnostic and treatment purposes. The key to using CTCs as predictive clinical biomarkers is their separation and enrichment. This work presents the use of a contactless dielectrophoresis (cDEP) device to investigate the frequency response of cells and calculate their area-specific membrane capacitance. This is the first demonstration of a cDEP device which is capable of operating between 10 and 100 kHz. Positive and negative dielectrophoretic responses were observed in red blood cells, macrophages, breast cancer, and leukemia cells. The area-specific membrane capacitances of MDA-MB231, THP-1 and PC1 cells were determined to be 0.01518 ± 0.0013, 0.01719 ± 0.0020, 0.01275 ± 0.0018 (F/m2), respectively. By first establishing the dielectrophoretic responses of cancerous cells within this cDEP device, conditions to detect and enrich tumor cells from mixtures with non-transformed cells can be determined providing further information to develop methods to isolate these rare cells