The authors would like to thank Boris Solomonov and the Virginia Commonwealth Innovation Machine Shop for help with rapid prototyping the device. The authors would also like to thank Cristian Romero-Fuentes of the Lewinski Group, Dr. Vitaliy Avrutin, Dr. Dmitry Pestov, and the Virginia Commonwealth Nanomaterials Core Characterization Facility for their help with particle characterization. This work was supported by startup funds provided to Dr. Lewinski by the College of Engineering at Virginia Commonwealth University.

Operators must wear personal protective equipment (e.g. lab coat, gloves, goggles) when performing steps 1, 2, 3, 5, and 6.
1. Preparation of Materials
<ol>
	<li>Prepare materials for system assembly and exposure to ensure repeatability.
	<ol>
		<li>Make sure to use new or 70% ethanol cleaned &#188;&#8221; inner diameter conductive tubing and &#188;&#8221; outer diameter connectors for the system assembly.</li>
		<li>Store test materials including filters, PIVEC components, tweezers, and particle powders in a well-controlled environment, with respect to the temperature and humidity, for at least 24 h prior to the experiment. 
		NOTE: The temperature should be near room temperature, approximately 20 &#176;C, with relative humidity less than 35%. This is very important to achieve repeatability between experiments.</li>
		<li>Prepare particle counters using isopropanol to clean parts and allow for the system warm-up according to the manufacturer recommendations, including the scanning mobility particle sizer (SMPS) and optical particle sizer (OPS) for measurement.</li>
	</ol>
	</li>
</ol>
2. Generation of Dry Aerosol
NOTE: Operators should perform aerosol generation in a fume hood.
<ol>
	<li>Assemble a system to generate dry aerosols 
	NOTE: The suspension of particles in gas or liquid should be appropriate for the modeled application and cell culture. The following method can be performed using a liquid-based aerosol. The design of the dry aerosol system is from Tiwari et al.37 A schematic of the dry dispersal system is shown in Figure 1.
	<ol>
		<li>Connect the ball valve to each end of the 4&#8221; 1/8 size threaded pipe, this will serve as the particle hopper. Connect 2&#8221; 1/8 size pipe to one valve.</li>
		<li>Weigh copper nanoparticles, in this study the mass concentration for each particle size was kept constant while determining the deposition efficiency. Approximately use 7.5 mg of 40 nm copper nanoparticles, 7 mg of 100 nm copper nanoparticles, and 13 mg of 800 nm copper nanoparticles per exposure. Place copper nanoparticles into the particle hopper through the open end. 
		NOTE: The amount of copper nanoparticles weighed will serve as the mass based administered concentration.</li>
		<li>Place a 3&#8221; piece of &#189;&#8221; outer diameter (OD) tubing around the 2&#8221; pipe and place a HEPA filter inside this short tubing such that the flow direction is through the ball valve.</li>
		<li>Connect the vacuum generator to other ball valve using threading. Connect vacuum generator to air tank by placing a 5/16&#8221; OD tubing into the push-to-lock connection. Use &#188;&#8221; OD tubing to connect the outlet of the vacuum generator to the experimental set-up by placing the tubing over the outlet of the vacuum generator.</li>
	</ol>
	</li>
	<li>Use of dry aerosol system to generate dry aerosol
	<ol>
		<li>Open the air tank by turning the main valve and allow the air flow to the system. Open the valve on the flow regulator on the air tank and set such that the flow through the system is equivalent to the desired settings on the vacuum pump.</li>
		<li>Open the ball valve closest to HEPA filter then open the ball valve closest to vacuum generator. Keep these open for approximately 3 s to allow particles to be pulled into the air stream.</li>
		<li>Close the ball valve closest to vacuum generator then close ball valve closest to HEPA filter. Allow air from the tank to flow for the duration of the experiment as necessary.</li>
		<li>Close main and regulator valves on air tank to stop the flow. Clean ball valves and vacuum generator using 70% ethanol. Autoclave metal pipes for sterilization.</li>
	</ol>
	</li>
</ol>
3. Deposition Efficiency Measurement using PIVEC
NOTE: Operators should perform aerosol exposures in a fume hood.
<ol>
	<li>Measure the deposition by collecting the copper nanoparticle aerosol generated in step 2.2 on a pre-weighed filter. Use the deposited dose, measured using the collected mass on the filter, and the administered dose, measured using the amount of weighed copper particles, to determine the deposition efficiency.
	<ol>
		<li>Keep 1.00 &#181;m pore glass fiber filters under low humidity conditions, described in 1.1.2, for at least 24 h prior to pre-exposure measurements. Weigh an unused filter three times and record the filter weights. Place the unused filter in a cell culture insert.</li>
		<li>Choose appropriate cell culture insert adapter (6 well or 24 well) for PIVEC to support the cell culture insert with the filter. Place the cell culture insert adapter piece on the top of the base of PIVEC, setting into place such that the base of the adapter piece is wider than the top.</li>
		<li>Use tweezers to place filter loaded cell culture insert within adapter piece. Place the top piece on top of adapter piece, settling into place such that the base of the top piece is wider than the top. Wrap PIVEC with a single layer of duct tape.</li>
		<li>Connect 37 mm cassette pieces on top and bottom of PIVEC by pushing into place. Place &#188;&#8221; barbed adapters into cassette inlet and outlet.</li>
		<li>Wrap the resistive heater around PIVEC such that the wires are at the base. Tape to secure.</li>
		<li>Wrap PIVEC with ~8 rounds of aluminum foil for insulation. Secure with tape.</li>
		<li>Connect 2&#8221; long piece of 1/2&#8221; outer diameter flexible tubing to the adapter on top of PIVEC. Remove porous tubing from sterile water and place within tubing on top of PIVEC.</li>
		<li>Place PIVEC within clamp on the ring stand and secure. Complete set-up with the vacuum pump, particle counters, and aerosol set-up. 
		NOTE: The number-based deposited dose can be determined only if particle counters are placed before the PIVEC and after the PIVEC on separate runs.</li>
		<li>Expose filters using step 2.2 of protocol and desired exposure time and flow rates, in this study an exposure time of 10 min at 0.5 LPM was used. Remove PIVEC from the set-up. Take out the cell culture insert and place the exposed filter in filter holder under low humidity conditions for at least 24 h prior to measurements.</li>
		<li>Clean PIVEC with 70% ethanol. Sterilize with ultraviolet light for at least 30 min prior to the next experiment.</li>
		<li>Weigh the exposed filter three times and record the filter weights. Place the exposed filter in a labeled filter holder for storage.</li>
	</ol>
	</li>
</ol>
4. Calculation of Deposited Dose and Deposition Efficiency
NOTE: Knowledge of the deposition is important for aerosol administration and interpretation of cellular response.
<ol>
	<li>Calculate deposition from mass-based measurements
	<ol>
		<li>Calculate the deposited mass on filters as the difference between the pre-exposure average weight and post-exposure average weight. This value is the mass-based deposited dose for the experiment.</li>
		<li>Use administered mass, madmin, and mass-based deposited dose determined in 4.1.1, mdep, to calculate the mass-based deposition efficiency, &#951;m, for the experiment. 
		<img alt="Equation" src="/files/ftp_upload/58916/58916eq1.jpg" /></li>
		<li>Average values from 4.1.1 and 4.1.2 for at least 3 experiments to determine deposition and deposition efficiency for PIVEC for particle size.</li>
	</ol>
	</li>
	<li>Calculate deposition from number-based measurements
	<ol>
		<li>Ensure that measurements with particle counters have been performed with counters after the PIVEC and to determine the particle concentration before the PIVEC. Integrate the particle concentration over time for the particle counter then integrate over particle diameter to determine the total particles measured.</li>
		<li>Calculate the deposited particle number as the difference between the particles administered and the particles measured post-PIVEC. This value is the number-based deposited dose for the experiment.</li>
		<li>Use administered particles, nadmin, number-based deposited dose, ndep, volumetric flow rate, V, and time, t, to calculate the number-based deposition efficiency, &#951;n, for the experiment. 
		<img alt="Equation" src="/files/ftp_upload/58916/58916eq2.jpg" /></li>
		<li>Average values from 4.2.2 and 4.2.3 for at least 3 experiments to determine deposition and deposition efficiency for PIVEC for particle size.</li>
	</ol>
	</li>
</ol>
5. Aerosol Exposure of Cells
NOTE: For the cell culture at the air-liquid interface the reader is referred to Blank et al.38 Operators should perform cell culture insert loading (steps 5.1.2-5.1.4) within a biosafety cabinet. Operators should perform aerosol exposures in a fume hood.
<ol>
	<li>Culture Cells at Air-Liquid Interface
	<ol>
		<li>Lift A549 cells from culture flask by adding trypsin-EDTA, 3 mL for a T75 flask or 1 mL for a T25 flask and incubate for 5 min at 37 &#176;C. Add 7 mL of complete media for a T75 flask or 4 mL of complete media for a T25 flask to flask and rinse flask wall with cell suspension to maximize the recovered cell number. Transfer the cell suspension to a sterile 15 mL conical tube then centrifuge cells at 800 x g for 3 min.</li>
		<li>Remove the supernatant containing trypsin-EDTA and resuspend the cell pellet in 10 mL of complete media. Remove 10 &#181;L of cell suspension and add to the hemocytometer. Count cells using a hemocytometer to determine the concentration and the total number of cells.</li>
		<li>Place 0.5 mL of complete media to each well within a 24 well plate. Place unused cell culture inserts in wells. Seed cell culture inserts on the apical side at a cell density near 1 x 105 cells/cm2 for cell types that grow at a rate near doubling per day. To seed A549 cells within a 24 well insert, seed cells at a density of 1 x 105 cells/cm2 by adding 35,000 cells to the cell culture insert. 
		NOTE: Cells with a slower growth rate can be seeded at an increased cell density.</li>
		<li>Add complete media to the apical side of cell culture insert to reach the final volume (for 24 well plate final volume is 0.25 mL).</li>
		<li>Culture for 7 days in submerged conditions, replacing media every 1-2 days. After 7 days, remove the apical media and culture for at least 1 day in ALI conditions, replacing only the basolateral media.</li>
	</ol>
	</li>
	<li>Assemble PIVEC 
	<ol>
		<li>Allow cells to equilibrate to the air-liquid interface for at least 24 h prior to exposure.</li>
		<li>Choose appropriate cell culture insert adapter for PIVEC to support the cell culture insert with the filter. Place cell culture insert adapter piece on top of PIVEC base, setting into place such that the base of the adapter piece is wider than the top. Add 4 mL of cell culture media to the well of the base of the PIVEC.</li>
		<li>Use tweezers to place the cell culture insert within adapter piece placed in step 5.2.3. Place top piece on top of adapter piece, settling into place such that the base of the top piece is wider than the top. Carefully, wrap PIVEC with a single layer of duct tape.</li>
		<li>Connect 37 mm cassette pieces on top and bottom of PIVEC, by pushing into place. Place &#188;&#8221; barbed adapters into cassette inlet and outlet.</li>
		<li>Wrap resistive heater around PIVEC such that the wires are at the base. Tape to secure.</li>
		<li>Wrap PIVEC with ~8 rounds of aluminum foil for insulation. Secure with tape.</li>
		<li>Connect short piece of 1/2&#8221; outer diameter flexible tubing to the adapter on the top of PIVEC. Remove porous tubing from sterile water and place within tubing on top of PIVEC.</li>
		<li>Place PIVEC within clamp on the ring stand and secure. Complete the set-up with the vacuum pump and aerosol set-up.</li>
	</ol>
	</li>
	<li>Expose Cells at ALI using the PIVEC
	<ol>
		<li>Use deposition efficiency determined in Step 2 to calculate the mass of particles to be aerosolized. Weigh appropriate mass and add to the aerosol system set up following step 2 within the fume hood.</li>
		<li>Expose cells by following step 2.2, in this study of biological endpoints the cells were exposed to approximately 3.5 mg of copper nanoparticles with a flow rate of 0.5 LPM and an exposure duration of 10 min. Control studies performed used humidified air to determine the influence of the air alone. Remove PIVEC from the set-up. Take out the cell culture insert, place in the sterile well plate and return to the CO2 incubator (37&#176;C, 5% CO2, 90% RH).</li>
		<li>Aspirate media from PIVEC. If performing additional experiments, rinse bottom of PIVEC with phosphate buffered solution then repeat step 5.1 and 5.2.</li>
		<li>Clean PIVEC with 70% ethanol when finished. Sterilize with ultraviolet light for at least 30 min prior to the next experiment.</li>
	</ol>
	</li>
	<li>Biological Assay Procedures 
	NOTE: Assays performed in this study were oxidative stress generation through the DCFH-DA assay and cytotoxicity through lactate dehydrogenase (LDH) release.
	<ol>
		<li>Dissolve 24.4 mg of DCFH-DA in 50 mL methanol to make 1 mM DCFH-DA solution. This solution can be stored at -20&#176;C for up to 4 months. Dilute 1 mM DCFH-DA solution by mixing 0.1 mL of 1 mM DCFH-DA solution with 9.9 mL HBSS to make 10 mL of 10 &#181;M DCFH-DA.</li>
		<li>Remove cell culture media and wash cell culture insert with approximately 1 mL of PBS. Add 0.5 mL of 10 &#181;M DCFH-DA solution to each well, replacing inserts when finished. Cover plate with aluminum foil to prevent photoactivation of the dye and return to 37&#176;C incubator for 1 h.</li>
		<li>Remove cells from the incubator and aspirate the DCFH-DA working solution from the wells. Add 0.5 mL HBSS to wells and replace cell culture inserts.</li>
		<li>Load the well plate into plate reader and measure baseline fluorescence using excitation/emission wavelengths of 485/530 nm. Remove plate from plate reader and load insert into PIVEC for exposure.</li>
		<li>Expose cells for desired exposure duration. Remove insert from PIVEC and return to well plate. Remove 50 &#181;L of basolateral fluid from well plate and place in white 96 well plate. Measure the fluorescence of DCF using excitation/emission wavelengths of 485/530 nm every 30 min post-exposure for 2 h.</li>
		<li>Let basolateral fluid equilibrate to room temperature for 20-30 min. Add 50 &#181;L of LDH assay solution, mixed following manufacturer protocol, to basolateral fluid from well plate and let react for 10 min. Add 25 &#181;L of stop solution to well. Read fluorescence of resorufin product using excitation/emission wavelengths of 560/590 nm.</li>
		<li>Remove additional basolateral fluid and repeat step 5.4.6 at 4 h and 24 h post-exposure.</li>
	</ol>
	</li>
</ol>
6 Statistical Methods
<ol>
	<li>Analysis of Biological Assay Data
	<ol>
		<li>Report ROS production as the fluorescence intensity increase of treated cells relative to baseline measurements. Report LDH activity as the fluorescence intensity increase of treated cells relative to untreated cells.</li>
		<li>Perform single factor ANOVA to determine statistical differences between data sets. Where appropriate, perform student t-tests at a value of significance of 0.05. Report data as the mean &#177; standard deviation of at least three exposure measurements.</li>
	</ol>
	</li>
</ol>

The affiliation of the authors is as shown on the cover page. The authors are financially supported by Virginia Commonwealth University, where the work was completed in Richmond, VA. The authors have sole responsibility for the writing and content of this paper. The authors declare that there are no competing interests.

Filter cassettes provide a simple, inexpensive method of collecting aerosols in the breathing zone; however, aerosol samples extracted from filters do not represent the entire aerosol (i.e. gases, volatiles, and particulates) and consequently limit the assessment of related biological effects. Using the initial design of the 37 mm filter cassette, the PIVEC is designed to maintain portability and mimic the in vivo deposition of particles from inhalation. The PIVEC is significantly smaller than current ALI exposu...

Personal sampling using in vitro techniques could provide comprehensive information regarding the biological effects of aerosols in the workplace.1 Exposures to contaminants in the air include exposures to the chemical itself, to the collected air samples, under submerged conditions where the gas is introduced to the cell suspension, intermittent exposures using a device such as a rocker, or direct exposures at the air-liquid interface (ALI).2 Many of these techniques are performed with cells grown in suspension or the collection of samples prior to the exposure, each of which can affect the toxicological study due to potential changes in the aerosol.3 To avoid these changes, the laboratory can be brought to the field using several in vitro ALI culture exposure systems that are used in literature,4,5,6,7,8,9,10,11,12,13 however, few are commercially available.8,9,12 These systems are often bulky, especially when including instruments to regulate the temperature and humidity of the cellular environment and the flow rate of the sample aerosol. By using the PIVEC, aerosol exposures can be performed outside of a traditional lab setting or within the breathing zone while mimicking inhalation conditions.
The determination of aerosol deposition in vitro is important to the investigation of health effects due to inhalation. The breathing zone, the area within 30 cm from the mouth and nose,14 is crucial for understanding the exposure to nanoparticles and for linking to the biological effects in the lungs.2 Often, the deposition on cells is defined as a deposition efficiency, the particles deposited onto and taken up by the cells divided by the particles administered to the system6,15 or on a mass basis of the same amounts.4,16 The current methods for measuring aerosols in the breathing zone are filter based, capturing particles over a given sampling period and using the filters to conduct further testing.17 Personal monitoring requires a small system that comes with the tradeoff of fewer samples.
There are many approaches to determine the health effects from exposure to an aerosol. The ALI model allows for the aerosol to be administered directly to the cells through the air as in a real exposure scenario, yet it is more cost-effective and less time intensive than in vivo studies while mimicking the air-liquid barriers such as the eyes, skin, and lungs. Lung cells grown at the ALI have the ability to generate a polarized barrier layer,18,19 which produces physiological traits that resemble the in vivo lung epithelium, including mucus and surfactant production in specific bronchial or alveolar cell lines, cilia beating,19 tight junctions,19,20 and cell polarization.18 Changes such as these can affect the cellular response measured in toxicity studies.21 In addition, ALI in vitro model results are often more sensitive than cells exposed via suspension models22 and are able to model acute in vivo inhalation toxicity.23,24 Therefore, an ALI exposure system that is able to perform measurements within the breathing zone is a natural next step.
By exposing the cells to aerosol directly at the source of emission, investigation of the effects of all gases, semi-volatile compounds, and particles involved in the mixture occurs. When the mixture is collected on a filter, the gases and volatile compounds are not captured and the whole mixture cannot be investigated. In addition, reconstitution of particles into a powder or a liquid suspension can lead to the aggregation or particle-fluid interactions, such as dissolution, in liquid suspension.25,26 When aerosol particles are added to the liquid, there is a higher potential for agglomeration,25,27 formation of a protein corona,28 or interaction with compounds in the liquid, which can affect deposition and influence the biological response.29,30
Exposure at the ALI is based on three main aerosol profiles, cloud settling, parallel flow, and perpendicular flow. Cloud settling, used by the Air-Liquid Interface Cell Exposure (ALICE),4 is a batch system where particles deposit through gravitational and diffusional settling as the aerosol is treated as one unit. Parallel flow, used by the Electrostatic Aerosol in vitro Exposure System (EAVES)5 and Multiculture Exposure Chamber (MEC) II,6 allows for deposition through the addition of Brownian motion through the flow profile. Perpendicular flow, used by a microsprayer,7 Nano Aerosol Chamber for In-Vitro Toxicity (NACIVT),11 and commercial ALI systems8,9,10,12, adds the impaction of particles within the deposition region. Many of these exposure systems are large and bulky, requiring excess systems for aerosol pre-conditioning, pumps for flow, or even heating chambers for incubation of cells. This large size decreases the portability of the system. Instead of sampling directly at the source of emission, these systems often have samples brought to the lab or model aerosols generated for analysis. The complexity of the emitted aerosol can be lost in translation from the field to the lab. The PIVEC is smaller than current systems, with an external surface area of approximately 460 cm2 and weighing only 60 grams, with thermal and humidity control incorporated into the system allowing for a highly portable device. The decreased size and weight allow the system to be worn or taken to the source of exposure, permitting direct sampling.
The large size of current exposure systems also decreases the ability to perform sampling to investigate spatial gradients in concentrations. This resolution is key when determining toxicological effects of many potential environmental and occupational hazards such as vehicular exhaust particulate matter or workplace activities where aerosolization occurs. Immediately post-emission, there becomes a spatial variance in particle concentration. This grows with time as the particles disperse throughout the atmosphere and these effects can change based on the ambient conditions, such as temperature, pressure, wind, and sun. Particles can begin to age and oxidize as well once emitted31,32 and dispersal rates are affected by the topography; higher concentrations will be found in canyons and tunnels, where dispersion effects are slowed, and lower concentrations can be found where there is a large area for dispersion.33 These changes in dispersion rates can have significant effects on human health and can be seen when comparing the number of asthmatic adults living in urban versus in rural settings.34 While many exposure systems provide multiple samples at once, multiple systems are necessary with an abundance of large equipment to perform spatial resolution.
By bringing the lab to the field, the time of analysis can be decreased by using the whole cell as a sensor. Following known biological mechanisms and endpoints can aid in the determination of the aerosol composition and size. Due to slow clearance methods, including mucociliary clearance, phagocytosis, and translocation, these particles are often interacting with cells for approximately days to weeks3 generating oxidative stress, inflammation, and even cell death. These biological endpoints can be the starting points for adverse outcome pathways for cardiovascular disease or chronic obstructive pulmonary disease. In addition, Wiemenn et al. performed an array of in vitro assays to compare with literature values for short term in vivo inhalation toxicity.35&#160;In vivo response was predicted with two of four positive results from testing cytotoxicity via lactate dehydrogenase release, oxidative stress from glutathione reduction and hydrogen peroxide formation and release, and inflammation potential from the tumor necrosis factor alpha gene. Out of ten nanosized metal oxides tested, six tested as active (titanium oxide, zinc oxide, and four different cerium oxide) using exposures in vitro with confirmation in vivo.
In order to study the effects of aerosols in an occupational setting, our lab developed the PIVEC for exposures in the field. Additionally, the PIVEC can be worn for personal sampling to monitor and investigate inhalation exposure like the 37 mm filter cassette36 or multiple systems can be used to achieve spatial resolution within a given area. In this protocol, the characterization and use of the PIVEC is discussed. After exposure, the biological effects are observed through cytotoxicity assays.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Scanning mobility particle sizer (SMPS)</td>
			<td>TSI, Inc.</td>
			<td>3910</td>
			<td>NanoSMPS</td>
		</tr>
		<tr>
			<td>Optical particle sizer (OPS)</td>
			<td>TSI, Inc.</td>
			<td>3330</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Pipe, 4&#34; Long</td>
			<td>McMaster-Carr</td>
			<td>4830K116</td>
			<td>Standard-Wall 304/304L, Threaded on Both Ends, 1/8 Pipe Size</td>
		</tr>
		<tr>
			<td>Brass Ball Valve with Lever Handle</td>
			<td>McMaster-Carr</td>
			<td>4112T12</td>
			<td>Compact High-Pressure Rating, 1/8 NPT Female</td>
		</tr>
		<tr>
			<td>Steel Pipe, 2&#34; Long</td>
			<td>McMaster-Carr</td>
			<td>7753K121</td>
			<td>Standard Wall, Threaded on One End, 1/8 Pipe Size</td>
		</tr>
		<tr>
			<td>HEPA filter</td>
			<td>GE Healthcare</td>
			<td>09-744-12</td>
			<td>HEPA-Cap Disposable Air Filtration Capsule</td>
		</tr>
		<tr>
			<td>Vacuum Generator</td>
			<td>PISCO USA</td>
			<td>VCH10-018C</td>
			<td></td>
		</tr>
		<tr>
			<td>PIVEC</td>
			<td>VCU</td>
			<td></td>
			<td>For design please contact authors</td>
		</tr>
		<tr>
			<td>Resistive heater</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34; barbed connectors</td>
			<td>Zefon International, Inc.</td>
			<td>459743</td>
			<td></td>
		</tr>
		<tr>
			<td>Porous tubing</td>
			<td>Scientific Commodities, Inc.</td>
			<td>BB2062-1814A</td>
			<td>Hydrophilic 10 um pores</td>
		</tr>
		<tr>
			<td>Battery power bank</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture insert</td>
			<td>Fisherbrand</td>
			<td>353095</td>
			<td>24 well plate insert</td>
		</tr>
		<tr>
			<td>Filter Forceps</td>
			<td>Fisherbrand</td>
			<td>09-753-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipette</td>
			<td>ThermoScientific</td>
			<td>13-711-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Fiber Filters</td>
			<td>SKC</td>
			<td>225-7</td>
			<td>Binder-Free Type AE Filter 37 MM 1.00 um pore</td>
		</tr>
		<tr>
			<td>Ultra Micro Balance</td>
			<td>A&#38;D</td>
			<td>BM-22</td>
			<td>Housed in environmental chamber</td>
		</tr>
		<tr>
			<td>37 mm filter cassette</td>
			<td>SKC</td>
			<td>225-3250</td>
			<td>Filter Cassette Blank, 37 mm, Clear Styrene</td>
		</tr>
		<tr>
			<td>Variable flow vacuum pump</td>
			<td>SKC</td>
			<td>220-5000TC</td>
			<td>AirChek TOUCH, 5 to 5000 mL/min</td>
		</tr>
		<tr>
			<td>Copper Particles</td>
			<td>U.S. Research Materials, Inc.</td>
			<td>US1090</td>
			<td>40 nm</td>
		</tr>
		<tr>
			<td>Copper Particles</td>
			<td>U.S. Research Materials, Inc.</td>
			<td>US1088</td>
			<td>100 nm</td>
		</tr>
		<tr>
			<td>Copper Particles</td>
			<td>U.S. Research Materials, Inc.</td>
			<td>US1117M</td>
			<td>800 nm</td>
		</tr>
	</tbody>
</table>

a new portable in vitro exposure cassette for aerosol sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

Occupational in vitro toxicology involves maintaining cellular viability while performing aerosol exposure. The PIVEC system is shown in Figure 2, including the temperature and humidity control and the worn PIVEC. The temperature was maintained using a battery-powered resistive heater and the aerosol humidified using increased natural humidification through a porous, wetted tube. In a controlled aerosol setting inside a laboratory, the PIVEC can be s...

Watch this Scientific Journal Video about A New Portable In Vitro Exposure Cassette for Aerosol Sampling at JoVE.com

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

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JoVE Journal

Environment

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Video: A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...