This research is supported by the CounterACT Program, National Institutes of Health (NIH), Office of the Director, and the National Institute of Environmental Health Sciences (NIEHS) Grant Number U54 ES015678 (CWW). SA is also supported by Children&#39;s hospital Colorado/Colorado School of Mines Collaboration Pilot Award #G0100394 and Children&#39;s Hospital Colorado Research Institue Pilot Award #G0100471.

1. Rat Cardiomyocyte Cultures
<ol>
	<li>All experiments were performed under protocols approved by the institutional animal care and use committee, IACUC.</li>
	<li>Obtain rat cardiomyocytes from the hearts (ventricles) of male rats (240-260 g) using methods described previously4. Briefly, anesthetize animals using an intraperitoneal injection of pentobarbital (100 mg/kg; confirm anesthesia by toe pinch method) and then remove hearts into 10.0 ml, 1 mM Ca2+ containing Krebs Ringer buffer, pH 7.4.</li>
	<li>Rinse the hearts 5-6x&#160;to remove blood and then switch to a Ca2+ free Krebs Ringer buffer containing 0.02% protease and 0.06% collagenase A (5.0 ml/heart). Incubate the hearts in this solution for 10-15 min&#160;at 37 &#176;C with occasional shaking.</li>
	<li>After 10-15 min, wash out the enzymatic solution with Ca2+ free Krebs Ringer buffer for an additional 5 min. Release the cells from the flaccid tissue using a 25 ml pipette by pipetting the suspension up and down several times.</li>
	<li>Separate the cells from tissue by filtering through a 70 &#956;m nylon mesh and allow them to settle in Krebs Ringer buffer containing 0.1 mM Ca2+. Suspend the cell pellet in 1.0 ml Krebs Ringer buffer containing 0.2 mM Ca2+.</li>
	<li>Carefully layer over 5.0 ml 60 &#956;g/ml bovine serum albumin, BSA, in 15 ml centrifuge tubes, to separate cardiomyocytes from nonmyocytes and allow them to settle for 30 min. The cardiomyocytes are heavier and move to the bottom of the tube. Remove carefully the supernatant cells and media. Repeat this step once to further purify the cells.</li>
	<li>Gradually transition by washing (in steps using 0.25 mM, 0.5 mM, 0.75 mM, and 1.0 mM Ca2+ containing buffer) the ventricular myocytes/cardiomyocytes to 1.0 mM Ca2+ containing buffer and resuspended in ACCT medium consisting of Dulbecco&#8217;s Modified Eagle Medium, DMEM containing 2 mg/ml BSA, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 100 IU/ml penicillin, and 100 &#956;g/ml&#160;streptomycin.</li>
	<li>Plate the cardiomyocytes in ACCT medium at a density of 100 to 150 cells/mm2 on 100 mm or 35 mm laminin coated plastic culture dishes or 40 x 22 mm glass coverslips precoated with laminin (1 &#956;g/cm2). After 1 hr, wash the dishes with 2.0 ml ACCT to remove cells that are not attached. Add 10.0 ml of fresh media and incubate the cells.</li>
</ol>
2. Differentiated Air-liquid Interface (ALI) Culture of Human Airway Epithelial Basal Cells
<ol>
	<li>Procure human tracheobronchial tissues from National Disease Research Interchange under Institutional Review Board approved protocols. Cell harvest and perform culture using a published procedure5,6. This procedure uses cell cultures as they are a precise model for human inhalation exposures to TICs, however, if needed one can isolate and grow at ALI mouse and/or rat airway epithelial cells7,8.</li>
	<li>Briefly, make small pieces (~&#188; inch) of the tissue after removing all connective tissue and lymph nodes. Wash tissue several times in Lactated Ringer solution. Add tissues to a 50 ml conical tube and add protease solution (1% Protease/0.01% DNase in minimal essential media, MEM, tissue to fluid ratio (1:10)).</li>
	<li>Rock the tissue at 4 &#176;C for overnight. End the dissociation of the tissue by adding 10% fetal bovine serum. Scrape the epithelial surface with a surgical scalpel and collect the cells by centrifugation (2,000 rpm for 10 min).</li>
	<li>Plate cells at passage one on collagen-coated snapwells in special ALI medium prepared as described in detail by Fulcher et al9. Culture for 5 days before changing to air-liquid&#160;interface (ALI) by removing apical media.</li>
	<li>Feed the cells using ALI media every alternate day and allow the cells to differentiate for additional 2-3 weeks (observe numerous beating cilia and mucus secretion) before performing exposures. Wash the apical surface with warm PBS along with media changes.</li>
</ol>
3. Chlorine Exposure
<ol>
	<li>Contain the chlorine exposure system (CES) inside a qualified chemical hood with an operational face velocity of 100 fpm that provides the necessary secondary containment to prevent exposure of personnel in the event of accidental chlorine leaks.</li>
	<li>Operate the system under slight positive pressure (0.5 inches of water). Dry air is fed at 15 L/min and an appropriate level of chlorine is fed to attain the desired final chlorine concentration. The chambers have a locking lid with 4 mini BCU locks and a low durometer silicone gasket to provide the pressure seal.</li>
	<li>Deliver the Cl2 mixture through a Mass Flow Controller. The CES uses a compressed gas cylinder containing 1.0% Cl2 in dry nitrogen.</li>
	<li>Regulate the dilution airflow using the custom designed control panel and similarly regulate the Cl2 concentration delivered to the exposure chambers. A low volume sampling pump pulls the exhaust from the chambers into a chlorine analyzer to monitor concentrations that are then recorded on a data logger connected to the analyzer.</li>
	<li>Measure the flow rates within the chambers prior to exposure using a flow meter to assure equal delivery and exhaust rates.</li>
	<li>Prepare the cell cultures for exposure by removing the supernatant media and adding fresh media (basolateral media in ALI cultures). Any pretreatments with agents could be performed at this time.</li>
	<li>Expose the ALI cultures of airway epithelial cells to Cl2 gas (50, 100, or 300 ppm for 30 min) in the two sealed polysulfone biocontainment chambers. The cardiomyocytes (submerged or confluent cultures on membranes) are exposed to 50 or 100 ppm Cl2 for 15 min.</li>
	<li>After exposure flush the chambers with air until the Cl2 level falls below 1 ppm and can be safely opened to remove cells (within 5 min).</li>
</ol>
4. Transepithelial Electrical Resistance (TER) Measurement
<ol>
	<li>Measure the TER of air-liquid-interface, ALI, cultures using an epithelial voltohmmeter with a pair of silver chloride &#8220;chopstick&#8221; electrodes.</li>
	<li>Equilibrate the chopstick electrode in the ALI media 15 min&#160;before use.</li>
	<li>Add warm media to the apical (1.0 ml) and basolateral (2.0 ml) surface and measure the TER using the chopstick electrodes of the voltohmmeter.</li>
	<li>Dip the shorter arm of the electrode in apical media and the longer arm in the basolateral media. Click the &#8216;measure&#8217; button on the voltohmmeter to evaluate the electrical resistance.</li>
	<li>The voltohmmeter has the option to measure ohms or k ohms. Subtract the resistance across a cell-free culture support from the resistance measured across each cell layer to yield the transepithelial resistance (TER).</li>
</ol>
5. Caspase Measurement
<ol>
	<li>Add fresh ALI media (2.0 ml) to the basolateral surface post exposure and incubate the cell at room temperature. Collect supernatant media at 4 and 24 hr.</li>
	<li>Measure caspase 3/7 activity in the media supernatants by using a commercial caspase 3/7 assay kit.</li>
</ol>
6. Western Blot and Immunocytochemistry
<ol>
	<li>Perform western blots using cell lysates as previously described10. Suspend the protein lysate (20 &#956;g) in 5x reduced sample buffer and boil for 5 min. Subject the protein lysate to SDS-PAGE (4-15%) and transfer the separated proteins to a nitrocellulose membrane by electrophoretic blotting.</li>
	<li>Block non-specific binding by incubating the membrane with 5% milk in wash buffer (PBS + 0.1% detergent) and probe the membranes with primary antibodies against SERCA2 or sarcomeric actin at 1:1,000 dilution, overnight at 4 &#176;C. Next, wash and incubate membranes with the respective peroxidase-conjugated secondary antibodies and develop for detection as described before10 using commercial peroxidase detection kit.</li>
	<li>For immunocytochemistry treat live cells grown on inserts or glass coverslips in 6-well plates with 0.4% Triton-X-100 in 10 mM sodium citrate buffer for 20 min&#160;after rinsing with PBS.</li>
	<li>Block nonspecific binding by treating the cells with 5% donkey serum for 20 min, and then incubate the cells with non-specific IgG or individual primary antibodies specific to sarcomeric actin or Ki-67.</li>
	<li>Wash the cells with PBS and incubate with fluorescent-conjugated secondary antibodies to detect the primary antibody and a nuclear stain (1 &#956;g/ml DAPI).</li>
	<li>Wash with PBS and mount coverslips using commercial mounting media.</li>
	<li>Visualize the staining using a fluorescent microscope.</li>
</ol>

<ol>
	<li>Mohan, 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=Acute+accidental+exposure+to+chlorine+gas:+clinical+presentation,+pulmonary+functions+and+outcomes.">Acute accidental exposure to chlorine gas: clinical presentation, pulmonary functions and outcomes.</a> Indian J Chest Dis Allied Sci. 52, 149-152 (2010).</li><li>Kose, 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=Myocardial+infarction,+acute+ischemic+stroke,+and+hyperglycemia+triggered+by+acute+chlorine+gas+inhalation.">Myocardial infarction, acute ischemic stroke, and hyperglycemia triggered by acute chlorine gas inhalation.</a> Am J Emerg Med. 27, 1021-1024 (2009).</li><li>Das, R., Blanc, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorine+gas+exposure+and+the+lung:+a+review.">Chlorine gas exposure and the lung: a review.</a> Toxicol Ind Health. 9, 439-455 (1993).</li><li>Claycomb, W. C., Palazzo, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+the+terminally+differentiated+adult+cardiac+muscle+cell:+a+light+and+scanning+electron+microscope+study.">Culture of the terminally differentiated adult cardiac muscle cell: a light and scanning electron microscope study.</a> Dev Biol. 80, 466-482 (1980).</li><li>Ahmad, 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=Bcl-2+suppresses+sarcoplasmic/endoplasmic+reticulum+Ca2+-ATPase+expression+in+cystic+fibrosis+airways:+role+in+oxidant-mediated+cell+death.">Bcl-2 suppresses sarcoplasmic/endoplasmic reticulum Ca2+-ATPase expression in cystic fibrosis airways: role in oxidant-mediated cell death.</a> Am J Respir Crit Care Med. 179, 816-826 (2009).</li><li>Ahmad, 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=Tissue+factor+signals+airway+epithelial+basal+cell+survival+via+coagulation+and+protease-activated+receptor+isoforms+1+and+2.">Tissue factor signals airway epithelial basal cell survival via coagulation and protease-activated receptor isoforms 1 and 2.</a> Am J Respir Cell Mol Biol. 48, 94-104 (2013).</li><li>Lam, H. C., 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=Isolation+of+mouse+respiratory+epithelial+cells+and+exposure+to+experimental+cigarette+smoke+at+air+liquid+interface.">Isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface.</a> J Vis Exp. (48), (2011).</li><li>Hosokawa, T., 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=Differentiation+of+tracheal+basal+cells+to+ciliated+cells+and+tissue+reconstruction+on+the+synthesized+basement+membrane+substratum+in+vitro.">Differentiation of tracheal basal cells to ciliated cells and tissue reconstruction on the synthesized basement membrane substratum in vitro.</a> Connect Tissue Res. 48, 9-18 (2007).</li><li>Fulcher, M. 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=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Methods Mol Med. , 107-183 (2005).</li><li>Ahmad, 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=SERCA2+regulates+non-CF+and+CF+airway+epithelial+cell+response+to+ozone.">SERCA2 regulates non-CF and CF airway epithelial cell response to ozone.</a> PloS One. 6, e10 (2011).</li><li>Martin, J. G., 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=Chlorine-induced+injury+to+the+airways+in+mice.">Chlorine-induced injury to the airways in mice.</a> Am J Respir Crit Care Med. 168, 568-574 (2003).</li><li>Evans, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorine:+state+of+the+art.">Chlorine: state of the art.</a> Lung. 183, 151-167 (2005).</li><li>Vliet, 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=Formation+of+reactive+nitrogen+species+during+peroxidase-catalyzed+oxidation+of+nitrite.+A+potential+additional+mechanism+of+nitric+oxide-dependent+toxicity.">Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity.</a> J Biol Chem. 272, 7617-7625 (1997).</li><li>Ahmad, 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=Lung+epithelial+cells+release+ATP+during+ozone+exposure:+signaling+for+cell+survival.">Lung epithelial cells release ATP during ozone exposure: signaling for cell survival.</a> Free Radic Biol Med. 39, 213-226 (2005).</li><li>Allen, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+system+for+exposure+of+cultured+cells+and+other+materials+to+ozone.">An automated system for exposure of cultured cells and other materials to ozone.</a> Inhal Toxicol. 15, 1039-1052 (2003).</li></ol>

The authors declare that they have no competing financial interests.

The most common type of acute toxic exposures occurs when one breathes a poisonous chemical into the lungs. These chemicals may also be quickly taken up in the bloodstream and may impact other organs such as brain and heart. Inhalation toxicity of various agents using animal models are studied and reported widely, however the mechanisms are less well understood. This is a major hurdle in developing effective therapies. Absence of in vitro exposure systems is a primary reason behind the lack of mechanistic insigh...

Exposure to toxic inhaled chemicals (TICs)/gases such as chlorine (Cl2) remains an ongoing health concern in accidental exposures as well as in their potential use as a chemical threat agent. Although the lungs are the primary target, organs such as heart and brain are also affected1-3. In vivo models are generally used for testing toxicity from TICs, but in vitro assays for toxicity assessment are simpler, faster and more cost effective. In vitro models also allow for extensive investigation of agent-cell interactions that may be difficult to evaluate in vivo. Such in vitro exposure systems are rare and moreover, in some conventional models where toxic agents are added to the culture medium in which cells are submerged, the properties of the agents can change due to interactions and binding to components in the medium. In such scenarios cell culture systems such as air-liquid&#160;interface (ALI) cultures of primary human airway epithelial cells, proposed here, that can be directly exposed to gaseous agents could be promising.
Epithelial cells lining the airway are the first lines of defense against inhaled toxic chemicals. The human airway epithelium forms a physical barrier between the lumen and the underlying cells in the lung and participates in the response of the lung. It produces a number of cytokines and other pro- and anti-inflammatory agents as well as secretes mucus/airway surface liquid (ASL) covering the epithelium. One of the limitations in conventional submerged in vitro culture systems is also that the ASL and mucus that cover the epithelial surface is removed or diluted. This does not reflect the physiological condition of lung epithelial cells that are exposed to air. Thus, an ideal in vitro system for TIC toxicity testing should replicate this architecture. There is great interest in developing rapid screening methods that predict in vivo toxicity. Epithelial cells grown at the ALI differentiate and have well-differentiated structures and functions compared to cells grown submerged and serve a superior model of the airways.
In this study, we describe the use of air-liquid-interface culture of human airway (tracheobronchial) epithelial cells for testing poisonous inhaled gas toxicity and compare it with a submerged cell culture of cardiomyocyte, hence studying another important target of toxicity.

<table border="0" cellpadding="0" cellspacing="0" width="1128">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Name</td>
			<td style="width:329px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Rats</td>
			<td style="width:329px;">Harlan Laboratories</td>
			<td>Sprague-Dawley&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Pentobarbital</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P3761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Chlorine</td>
			<td style="width:329px;">AirGas, Inc</td>
			<td style="width:147px;">X02NI99CP163LS1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Caspase 3/7 kit&#160;</td>
			<td style="width:329px;">Promega</td>
			<td style="width:147px;">G8091</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Epithelial voltohmmeter and chopstick electrode</td>
			<td style="width:329px;">World Precision Instruments</td>
			<td style="width:147px;">EVOM and STX2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Snapwell inserts</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">07-200-708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">70 micron nylon cell strainer</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">#352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysulfone biocontainment chambers&#160;</td>
			<td style="width:329px;">BCU, Allentown Cage Equipment</td>
			<td style="width:147px;">BCU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:329px;">Life technologies</td>
			<td style="width:147px;">12491-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sarcomeric actin antibody</td>
			<td style="width:329px;">Abcam Cambridge, MA</td>
			<td style="width:147px;">ab28052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SERCA2 antibody</td>
			<td style="width:329px;">&#160;Affinity Bioreagents, Golden, CO</td>
			<td style="width:147px;">MA3-9191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ki-67 antibody</td>
			<td style="width:329px;">&#160;Dako, Carpinteria, CA</td>
			<td style="width:147px;">M7248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-488-conjugated secondary antibody</td>
			<td style="width:329px;">&#160;Invitrogen, Grand Island, NY</td>
			<td style="width:147px;">A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">BSA</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Carnitine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Taurine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Creatinine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Krebs Ringer Buffer</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">K4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagenase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DNAase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Lactated Ringer solution</td>
			<td style="width:329px;">Abott Laboratories</td>
			<td style="width:147px;">7953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Donkey serum</td>
			<td style="width:329px;">Fisher Scientific</td>
			<td style="width:147px;">017-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PBS, phosphate buffered saline</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4-15% SDS-PAGE gels</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">456-1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dergent, Tween&#160;</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Peroxidase detection kit</td>
			<td style="width:329px;">Pierce</td>
			<td align="right" style="width:147px;">3402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">DAPI</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Mounting media, Fluormount G</td>
			<td style="width:329px;">eBiosciences</td>
			<td style="width:147px;">00-4958-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium citrate</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">71497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MEM</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">M8028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Laminin</td>
			<td style="width:329px;">BD biosciences</td>
			<td style="width:147px;">354259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/Streptomycin</td>
			<td style="width:329px;">Life Technologies</td>
			<td style="width:147px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FBS</td>
			<td style="width:329px;">Gibco</td>
			<td style="width:147px;">200-6140AJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cell culture model for toxic inhaled chemical testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

Primary rod shaped cardiomyocytes attach on laminin matrices and spread and differentiate into confluent cultures (Figure 1A and its inset). These cells were further characterized on the basis of sarcomeric actin and SERCA2 expression (Figures 1B and 1C). Rat cardiomyocytes are highly susceptible to chlorine toxicity as 15 min exposure to 100 ppm chlorine caused extensive cell rounding and death in submerged cultures and disruption of confluent layers on cells grown on l...

Watch this Scientific Journal Video about In vitro Cell Culture Model for Toxic Inhaled Chemical Testing at JoVE.com

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

in-vitro-cell-culture-model-for-toxic-inhaled-chemical-testing

Research

JoVE Journal

Bioengineering

10.6K Views.  University of Colorado. This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow impo...