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1.  Real-time cell viability assays using a new anthracycline derivative DRAQ7® 
The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a non-invasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be non-toxic to a panel of cancer cell lines grown continuously for up to 72 hours and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation and drug-induced cytotoxicity. The overall responses to anti-cancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨm) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multi-parameter and kinetic fingerprint of anti-cancer drug action.
PMCID: PMC3558543  PMID: 23165976
DRAQ7; real-time assays; cell viability; drug; cytotoxicity; DNA damage response; cell cycle; microfluidic; cytometry
2.  Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry 
Proceedings of SPIE  2013;8615:10.1117/12.2001474.
Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (μFCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The μFCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the μFCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that μFCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨm) in relation to DNA content (cell cycle phase) in live tumor cells.
PMCID: PMC3877312  PMID: 24386542
microfluidics; Lab-on-a-Chip; flow cytometry; cell cycle; apoptosis; programmed cell death; cancer
3.  Kinetic viability assays using DRAQ7 probe 
Cell death within cell populations is a stochastic process where cell-to-cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end-point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death-inducing signal There is a need therefore for rapid and high-throughput bioassays capable to continuously track viability of individual cells from the time of encountering a stress signal up to final stages of their demise. In this context, a new anthracycline derivative DRAQ7 is gaining increasing interest as an easy to use marker capable of long-term monitoring of cell death in real-time. This novel probe neither penetrates the plasma membrane of living cells nor does it affect cells susceptibility to the death inducing agents. However when the membrane integrity is compromised DRAQ7 enters cells undergoing demise and binds readily to nuclear DNA to report cell death. Here, we provide three sets of protocols for viability assays using DRAQ7 probe. The first protocol describes the innovative use of single color DRAQ7 real-time assay to dynamically track cell viability. The second protocol outlines a simplified end-point DRAQ7 staining approach. The final protocol highlights the real-time and multiparametric apoptosis assay utilizing DRAQ7 dye concurrently with tetramethylrhodamine methyl ester (TMRM), the mitochondrial trans-membrane electrochemical potential (ΔΨm) sensing probe.
PMCID: PMC3873765  PMID: 23835805
4.  Activities of occupational physicians for occupational health services in small-scale enterprises in Japan and in the Netherlands 
Occupational health service (OHS) for small-scale enterprises (SSEs) is still limited in many countries. Both Japan and the Netherlands have universal OHS systems for all employees. The objective of this survey was to examine the activities of occupational physicians (OPs) in the two countries for SSEs and to investigate their proposals for the improvement of service.
Questionnaires on types and sizes of the industries they serve, allocation of service hours (current and desired), sources of information for occupational health activities etc. were mailed in 2006 to 461 and 335 Japanese and Dutch OPs, respectively, who have served in small- and medium-scale enterprises. In practice, 107 Japanese (23%) and 106 Dutch physicians (32%) replied, respectively.
Results and Conclusions
Total service time per month was longer for OPs in the Netherlands than OPs in Japan. Japanese OPs spent more hours for health and safety meetings, worksite rounds, and prevention of overwork-induced ill health (14–16% each). Dutch OPs used much more hours for the guidance of absent workers (48%). Thus, service conditions were not the same for OPs in the two countries. Nevertheless, both groups of OPs unanimously considered that employers are the key persons for the improvement of OHS especially in SSEs and their education is important for better OHS. The conclusions should be taken as preliminary, however, due to study limitations including low response rates in both groups of physicians.
PMCID: PMC2836247  PMID: 20130904
Education; Employer; Occupational physician; Occupational health services; Small-scale enterprises
5.  Sympathetic Hyperactivity during Hypothalamic Stimulation in Spontaneously Hypertensive Rats 
Journal of Clinical Investigation  1978;62(3):642-648.
To determine whether sympathetic hyperactivity of hypothalamic origin contributes to keep blood pressures high in spontaneous hypertension, aortic pressures and sympathetic nerve spike potentials were recorded during electrical stimulation of the posterior hypothalamus in urethane-anesthetized normotensive or hypertensive rats. Basal sympathetic nerve activity was higher in spontaneously hypertensive rats than in either normotensive or deoxycorticosterone acetate-salt hypertensive ones even before stimulation began. Blood pressure elevations produced by hypothalamic stimulation were always preceded by substantial increases in amplitude and rate of neural firing. Changes in amplitude could not be quantified, but rates of neural firing accelerated much more in spontaneous hypertensives than in normotensives during stimulation with 50- and 100-μA currents. Similar differences between deoxycorticosterone acetate-salt hypertensives and either normotensives or spontaneous hypertensives were not statistically significant. Nerve activity invariably became quiescent immediately after hypothalamic stimulation was discontinued, and recovery from this poststimulatory inhibition was faster in spontaneously hypertensive than in normotensive rats. Although spontaneous hypertensives generally also had stronger pressor responses to various sympathomimetic stimuli, responses to hypothalamic stimulation were enhanced to a greater extent than those to either norepinephrine or sympathetic nerve stimulation. Because this selectivity indicates participation of mechanisms other than augmented cardiovascular reactivity, further enhancement of responsiveness to hypothalamic stimuli was attributed to the associated increase in sympathetic nerve firing. These results are in accord with the hypothesis that the blood pressure elevation in rats with established spontaneous hypertension is a result, at least in part, of sympathetic hyperactivity emanating from the posterior hypothalamus.
PMCID: PMC371810  PMID: 690189

Results 1-5 (5)