The authors are grateful to all the participants and their families, as well as to the teaching and administrative personnel of the participating primary schools in Cyprus and Greece. The study was financed by the European Union LIFE Project MEDEA (LIFE16 CCA/CY/000041).

<ol>
	<li>Alami, H., Gagnon, M. P., Fortin, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+health+and+the+challenge+of+health+systems+transformation.">Digital health and the challenge of health systems transformation.</a> mHealth. 3, 31 (2017).</li><li>Dunn, J., Runge, R., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wearables+and+the+medical+revolution.">Wearables and the medical revolution.</a> Personalized Medicine. 15 (5), 429-448 (2018).</li><li>Bietz, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+in+the+use+of+personal+health+data+for+health+research.">Opportunities and challenges in the use of personal health data for health research.</a> Journal of the American Medical Informatics Association. 23, 42-48 (2016).</li><li>Coughlin, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recall+bias+in+epidemiologic+studies.">Recall bias in epidemiologic studies.</a> Journal of Clinical Epidemiology. 43 (1), 87-91 (1990).</li><li>Munos, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobile+health:+The+power+of+wearables,+sensors,+and+apps+to+transform+clinical+trials.">Mobile health: The power of wearables, sensors, and apps to transform clinical trials.</a> Annals of the New York Academy of Sciences. 1375 (1), 3-18 (2016).</li><li>Reeder, B., David, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+at+hand:+A+systematic+review+of+smart+watch+uses+for+health+and+wellness.">Health at hand: A systematic review of smart watch uses for health and wellness.</a> Journal of Biomedical Informatics. 63, 269-276 (2016).</li><li>Trifan, A., Oliveira, M., Oliveira, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+sensing+of+health+outcomes+through+smartphones:+Systematic+review+of+current+solutions+and+possible+limitations.">Passive sensing of health outcomes through smartphones: Systematic review of current solutions and possible limitations.</a> JMIR mHealth and uHealth. 7 (8), 12649 (2019).</li><li>Rodgers, M. M., Alon, G., Pai, V. M., Conroy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wearable+technologies+for+active+living+and+rehabilitation:+current+research+challenges+and+future+opportunities.">Wearable technologies for active living and rehabilitation: current research challenges and future opportunities.</a> Journal of Rehabilitation and Assistive Technologies Engineering. 6, 2055668319839607 (2019).</li><li>Huberty, J., Ehlers, D. K., Kurka, J., Ainsworth, B., Buman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+three+wearable+sensors+for+24+hour+monitoring+in+middle-aged+women.">Feasibility of three wearable sensors for 24 hour monitoring in middle-aged women.</a> BMC Women's Health. 15, 55 (2015).</li><li>Schall, M. C., Sesek, R. F., Cavuoto, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barriers+to+the+adoption+of+wearable+sensors+in+the+workplace:+A+survey+of+occupational+safety+and+health+professionals.">Barriers to the adoption of wearable sensors in the workplace: A survey of occupational safety and health professionals.</a> Human Factors. 60 (3), 351-362 (2018).</li><li>Henriksen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fitness+trackers+and+smartwatches+to+measure+physical+activity+in+research:+Analysis+of+consumer+wrist-worn+wearables.">Using fitness trackers and smartwatches to measure physical activity in research: Analysis of consumer wrist-worn wearables.</a> Journal of Medical Internet Research. 20 (3), 9157 (2018).</li><li>Kouis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MEDEA+childhood+asthma+study+design+for+mitigation+of+desert+dust+health+effects:+Implementation+of+novel+methods+for+assessment+of+air+pollution+exposure+and+lessons+learned.">The MEDEA childhood asthma study design for mitigation of desert dust health effects: Implementation of novel methods for assessment of air pollution exposure and lessons learned.</a> BMC Pediatrics. 21, 13 (2021).</li><li>Kouis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+wearable+sensors+to+assess+compliance+of+asthmatic+children+in+response+to+lockdown+measures+for+the+COVID-19+epidemic.">Use of wearable sensors to assess compliance of asthmatic children in response to lockdown measures for the COVID-19 epidemic.</a> Scientific Reports. 11, 5895 (2021).</li><li>Arigo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+and+future+of+digital+health+in+the+field+of+behavioral+medicine.">The history and future of digital health in the field of behavioral medicine.</a> Journal of Behavioral Medicine. 42 (1), 67-83 (2019).</li><li>Fuller, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+and+validity+of+commercially+available+wearable+devices+for+measuring+steps,+energy+expenditure,+and+heart+rate:+Systematic+review.">Reliability and validity of commercially available wearable devices for measuring steps, energy expenditure, and heart rate: Systematic review.</a> JMIR mHealth and uHealth. 8 (9), 18694 (2020).</li><li>Majumder, S., Mondal, T., Deen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wearable+sensors+for+remote+health+monitoring.">Wearable sensors for remote health monitoring.</a> Sensors. 17 (1), 130 (2017).</li><li>Beukenhorst, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engagement+and+participant+experiences+with+consumer+smartwatches+for+health+research:+Longitudinal,+observational+feasibility+study.">Engagement and participant experiences with consumer smartwatches for health research: Longitudinal, observational feasibility study.</a> JMIR mHealth and uHealth. 8 (1), 14368 (2020).</li><li>Galarnyk, M., Quer, G., McLaughlin, K., Ariniello, L., Steinhubl, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usability+of+a+wrist-worn+smartwatch+in+a+direct-to-participant+randomized+pragmatic+clinical+trial.">Usability of a wrist-worn smartwatch in a direct-to-participant randomized pragmatic clinical trial.</a> Digital Biomarkers. 3 (3), 176-184 (2019).</li><li>de Lima, A. L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+wearable+sensor+deployment+in+Parkinson's+patients:+The+Parkinson@+home+study+protocol.">Large-scale wearable sensor deployment in Parkinson's patients: The Parkinson@ home study protocol.</a> JMIR Research Protocols. 5 (3), 5990 (2016).</li><li>Steinle, S., Reis, S., Sabel, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+human+exposure+to+air+pollution-Moving+from+static+monitoring+to+spatio-temporally+resolved+personal+exposure+assessment.">Quantifying human exposure to air pollution-Moving from static monitoring to spatio-temporally resolved personal exposure assessment.</a> Science of the Total Environment. 443, 184-193 (2013).</li><li>Dias, D., Tchepel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+of+human+exposure+to+air+pollution+in+the+urban+environment:+a+GPS-based+approach.">Modelling of human exposure to air pollution in the urban environment: a GPS-based approach.</a> Environmental Science and Pollution Research. 21 (5), 3558-3571 (2014).</li><li>Chen, W., Kao, K., Chang, Y., Chang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RSSI-based+distributed+real-time+indoor+positioning+framework.">An RSSI-based distributed real-time indoor positioning framework.</a> 2018 IEEE International Conference on Applied System Invention (ICASI). , 1288-1291 (2018).</li><li>Nagah Amr, M., El Attar, H. M., Abd El Azeem, M. H., El Badawy, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+indoor+positioning+technique+based+on+a+novel+received+signal+strength+indicator+distance+prediction+and+correction+model.">An enhanced indoor positioning technique based on a novel received signal strength indicator distance prediction and correction model.</a> Sensors. 21 (3), 719 (2021).</li><li>Lancioni, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upgraded+smartphone-based+program+for+leisure+and+communication+of+people+with+intellectual+and+other+disabilities.">An upgraded smartphone-based program for leisure and communication of people with intellectual and other disabilities.</a> Frontiers in Public Health. 6, 234 (2018).</li><li>Lancioni, G. 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The authors have no conflicts of interest to declare.

Wearable sensors are useful tools that allow the continuous and non-invasive monitoring of health parameters and patient behavior. Commercial smartwatches, which are equipped with a variety of sensors, provide a promising alternative to traditional data collection methods, and their use in clinical and public health research is only expected to rise as a result of increased variety and quality of built-in sensors, stronger academic-industry partnerships, and reductions in retail prices14. In this ...

Digital health technology applications and wearable sensors enable non-invasive and cost-effective patient monitoring both in healthcare and home settings1. At the same time, the large amount of data collected and the availability of wearable-based analytic platforms enable the development of algorithms for automated health event prediction, prevention, and intervention for a wide range of acute and chronic diseases2. Commercially available wearable sensors, primarily used for fitness tracking, are also increasingly being used by medical professionals in public health research and represent a promising tool for multimodal and continuous data collection under real-life conditions3. More importantly, though, unbiased data collection from wearables sensors allows researchers to overcome the challenges of recall bias that characterize traditional data collection methods such as interviews and diaries4.
However, for the purposes of clinical trials or other population studies, data accuracy, reliability, and integrity are essential. In addition, the credibility of the collected data may also be affected by several other parameters, such as age-group applicability as well as the memory capacity and energy efficiency of the device5. Recent systematic reviews of laboratory and field-based studies with limited numbers of participants have generally confirmed the applicability of commercial smartwatches for activity, heart rate, seizure, and behavior monitoring, although the reviews have also demonstrated poor suitability for elderly users, as well as battery, memory, and data quality limitations6,7. These limitations may be further amplified in larger population studies under real-life conditions where additional parameters such as inconsistent internet connectivity, device comfort, and incorrect smartwatch use come into play8. Specifically, appearance and inconvenience are significant barriers to wearing sensors daily9, while concerns relating to privacy and confidentiality issues may affect recruitment in studies involving wearable sensors10. Concerning the applicability of commercial smartwatches and fitness trackers for measuring physical activity in research studies, a recent study by Henriksen et al. suggested that the selection of an appropriate device for a particular study should not only be based on the available embedded sensors but rather also take into account validation and previous use in research, appearance, battery life, robustness, water resistance, connectivity, and usability11.
For the purposes of this study, we present a protocol to improve on the challenges encountered during the LIFE MEDEA project, an intervention study for the mitigation of the health effects of desert dust storms12. The study involved two distinct population groups: asthmatic children aged 6-11 years and elderly patients with atrial fibrillation (AF). Both groups were equipped with a commercial smartwatch for the assessment of physical activity (using a heart rate monitor, pedometer, and accelerometer) and location (using GPS signals to locate individuals in indoor &#34;at home&#34; or outdoor microenvironments). The participants were required to wear the smartwatch daily, and data were transmitted via a wireless network to a centrally administered data collection platform via the data collection application for the near real-time assessment of compliance. Additional details on the smartwatch and system setup are provided in a previous study13. During the first year of the project implementation, several technical and real-life challenges relating to the device emerged, which affected recruitment, the compliance of participants in wearing the device daily, and the completeness of the collected data. Some challenges were population-specific, such as the requirement of school administrators and many parents that the children wearing the smartwatches should not have access to standard smartwatch features, such as gaming, internet browser, camera, and audio-recording applications. Other challenges were technical in nature, such as loss of GPS signal, especially in indoor environments, and internal smartwatch settings interfering with the data collection application. A detailed overview of the main challenges identified as well as a brief description of their implications and solutions are presented in Table 1.
In this study, we suggest simple, cost-effective, and off-the-shelf solutions to improve user compliance, data quality, and data completeness in population studies employing wearable sensors and provide the relevant protocols. In addition, we demonstrate the data completeness improvements from the implementation of such protocols using representative results from the study13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>APK Extractor</td>
			<td>Meher</td>
			<td>Version 4.21.08</td>
			<td>Application</td>
		</tr>
		<tr>
			<td>Charger/Adaptor with data cable</td>
			<td>Jiangsu Chenyang Electron Co. Ltd</td>
			<td>C-P17</td>
			<td>Charger</td>
		</tr>
		<tr>
			<td>Embrace application</td>
			<td>EmbraceTech LTD</td>
			<td>Version 1.5.4</td>
			<td>Application</td>
		</tr>
		<tr>
			<td>LEMFO LF25 Smartwatch</td>
			<td>Shenzhen domino Times Technology Co. Ltd</td>
			<td>DM368 Plus</td>
			<td>Smartwatch</td>
		</tr>
		<tr>
			<td>Lock App - Smart App Locker</td>
			<td>ANUJ TENANI</td>
			<td>Version 4.0</td>
			<td>Application</td>
		</tr>
		<tr>
			<td>Macrodroid-Device Automation</td>
			<td>ArloSoft</td>
			<td>Version 5.5.2</td>
			<td>Application</td>
		</tr>
		<tr>
			<td>Xiaomi Redmi 6A</td>
			<td>Xiaomi</td>
			<td>M1804C3CG</td>
			<td>Smartphone</td>
		</tr>
	</tbody>
</table>

setup of consumer wearable devices for exposure and health monitoring in population studies

Wearable sensors, which are often embedded in commercial smartwatches, allow for continuous and non-invasive health measurements and exposure assessments in clinical studies. Nevertheless, the real-life application of these technologies in studies involving a large number of participants for a significant observation period may be hindered by several practical challenges. 
In this study, we present a modified protocol from a previous intervention study for the mitigation of health effects from desert dust storms. The study involved two distinct population groups: asthmatic children aged 6-11 years and elderly patients with atrial fibrillation (AF). Both groups were equipped with a smartwatch for the assessment of physical activity (using a heart rate monitor, pedometer, and accelerometer) and location (using GPS signals to locate individuals in indoor "at home" or outdoor microenvironments). The participants were required to wear the smartwatch equipped with a data collection application on a daily basis, and data were transmitted via a wireless network to a centrally administered data collection platform for the near real-time assessment of compliance. 
Over a period of 26 months, more than 250 children and 50 patients with AF participated in the aforementioned study. The main technical challenges identified included restricting access to standard smartwatch features, such as gaming, internet browser, camera, and audio recording applications, technical issues, such as loss of GPS signal, especially in indoor environments, and the internal smartwatch settings interfering with the data collection application.
The aim of this protocol is to demonstrate how the use of publicly available application lockers and device automation applications made it possible to address most of these challenges in a simple and cost-effective way. In addition, the inclusion of a Wi-Fi received signal strength indicator significantly improved indoor localization and largely minimized GPS signal misclassification. The implementation of these protocols during the roll-out of this intervention study in the spring of 2020 led to significantly improved results in terms of data completeness and data quality.

The protocol describes simple and cost-effective solutions to real-life challenges affecting recruitment, compliance, and data quality in population studies employing wearable sensors. The steps described here allowed for the successful setup of a consumer wearable device for exposure and health monitoring in a large population study involving children with asthma and adults with atrial fibrillation. Figure 6 provides a graphical overview of the provided protocols and illustrates the key ste...

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Setup of Consumer Wearable Devices for Exposure and Health Monitoring in Population Studies

Commercial smartwatches equipped with wearable sensors are increasingly being used in population studies. However, their utility is often constrained by their limited battery duration, memory capacity, and data quality. This report provides examples of cost-effective solutions to real-life technical challenges encountered during studies involving asthmatic children and elderly cardiac patients.

Wearable sensors, which are often embedded in commercial smartwatches, allow for continuous and non-invasive health measurements and exposure assessments ...

setup-consumer-wearable-devices-for-exposure-health-monitoring

Research

JoVE Journal

Bioengineering

2.3K Views.  University of Cyprus. Commercial smartwatches equipped with wearable sensors are increasingly being used in population studies. However, their utility is often constrained by their limited battery duration, memory capacity, and data quality. This report provides examples of cost-effective solutions to real-life technical challenges encountered during studies involving asthmatic children and elderly cardiac patients.

Video: Setup of Consumer Wearable Devices for Exposure and Health Monitoring in Population Studies

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...