This work is funded by R.J. Reynolds Tobacco Company (RJRT) under a collaborative research agreement with Wake Forest University School of Medicine. G.L. Prasad is a full time employee of RJRT.

NOTE: Written Informed consent to do this study was obtained at a local clinical research unit under IRB approval, per Good Clinical Practices. Processing of blood, isolation of PBMCs and other cell culture experiments are performed under sterile conditions, using microbiologically sterile supplies and reagents.
1. WS-CM Preparation
<ol>
	<li>Generate WS-CM as previously described12.
	<ol>
		<li>Prepare WS-CM by passing smoke from four 3R4F reference cigarettes through Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red using the following smoking regimen: 35-60-2, puff volume in ml, puff interval in sec, and puff duration in sec, respectively. Each preparation generates a 20 ml sample.</li>
	</ol>
	</li>
	<li>Label tube(s) with date, time completed, and cigarette name and number. Store the 500 &#181;l aliquots at -80 &#176;C immediately after smoking is completed.</li>
	<li>Analyze aliquots of frozen WS-CM to determine the levels of nicotine, tobacco specific nitrosamines, and polycyclic aromatic hydrocarbons as previously described12.</li>
</ol>
2. Isolation of PBMCs
<ol>
	<li>Collect fresh blood from healthy donors (who are non-consumers of tobacco products) Isolate PBMCs from fresh blood as described below under a separate approval from Wake Forest Baptist Health IRB15.
	<ol>
		<li>Prior to the arrival of the blood bag, have a 500 ml bottle, scissors, isolation buffer, and Dulbecco&#8217;s phosphate buffered saline (DPBS) (RT) ready in a biosafety level 2 (BSL-2) cell culture hood. Isolation buffer must be protected from light.</li>
		<li>Hold the blood bag upside down and cut the tube just below where it has been clamped leaving at least 3 cm of tubing.</li>
		<li>Remove the cap from the 500 ml bottle and hold the tube above the bottle opening. Pick up the blood bag and invert it to allow the blood to flow freely from the bag into the bottle until the bag is empty. Allow the blood to flow onto the inside wall of the bottle versus straight down as to avoid creating bubbles.</li>
		<li>Pour isolation buffer into the bottle at a 1:5 ratio of isolation buffer to blood. Cap the bottle tightly and gently invert it, end-over-end, 10 times. Leave the bottle in the cell culture hood to incubate with lights off, for 1 hr at RT.</li>
		<li>A light, straw-colored layer will build up above the blood. Remove this layer using a 25 ml serological pipette into 50 ml conical tubes. The collected amount may vary from 50 - 300 ml, depending on the subject who donated blood.</li>
		<li>Centrifuge the tubes at 200 x g for 10 min at RT.</li>
		<li>Aspirate the translucent supernatant, leaving the dark blood-colored pellet. The pellet will be loose but viscous.</li>
		<li>Pipette 3 ml of isolation buffer into 15 ml conical tubes.</li>
		<li>To the resuspend pellet, add 20 ml of DPBS for every 50 ml of straw-colored liquid collected in step 2.1.5. Vortex to mix thoroughly, and consolidate the resuspended liquid from multiple tubes. This liquid contains suspended blood cells.</li>
		<li>With a transfer pipet, transfer 5 ml of the suspended blood cells onto 3 ml of isolation buffer in step 2.1.8. Tilt the 15 ml conical tube that contains the cell suspension slowly and gently to create two separate layers. Centrifuge the tubes at 400 x g for 40 min at RT with minimal acceleration and without brake.</li>
		<li>Use a transfer pipet to remove the resulting cloudy middle layer (buffy coat) containing PBMCs into a 50 ml conical tube. Avoid drawing other clear layers below it. Transfer no more than 25 ml into each 50 ml conical tube.</li>
		<li>Add 25 ml of cold running buffer (or more to fill the entire remaining volume of the 50 ml conical tube) to wash the cells. Centrifuge the cells at 400 x g for 10 min at 4 &#176;C.</li>
		<li>Resuspend the pellet with 10 ml running buffer. This contains PBMCs. Count the cells and use immediately or place on ice for freezing.</li>
	</ol>
	</li>
</ol>
3. Freezing and Thawing the PBMCs
<ol>
	<li>Centrifuge the PBMCs that were collected in step 2.1.13 for 10 min at 400 x g.</li>
	<li>Fill the freezing container with isopropyl alcohol per the manufacturer&#8217;s instructions. CAUTION: Isopropyl alcohol is flammable and acutely toxic.</li>
	<li>Resuspend the pellet with RPMI 1640 medium (4 &#176;C) containing 20% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). This is RPMI 1640 freezing medium. Resuspend the pellet with an amount of freezing medium that will result in a suspension having about 5 x 107 cells/ml. The number of cells available for freezing will vary.</li>
	<li>Dispense 1 ml aliquots of cell suspension into 2 ml cryotubes and place the cryotubes in the freezing container. Place the freezing container with cryotubes in a freezer at -80 &#176;C to store O/N and then remove the cryotubes and transfer to store in a cryogenic freezer between -150 &#176;C to -190 &#176;C.</li>
	<li>Remove the cryotube from cryogenic storage and thaw it rapidly with gentle agitation in a water bath at 37 &#176;C.</li>
	<li>Immediately transfer the thawed PBMCs in the cryotube into a 15 ml conical tube with 10 ml RPMI 1640 complete medium (4 &#176;C) containing 10% FBS, 1% Pen/Strep and 1% L-glutamine. The contents of the thawed cryotube should be transferred as soon as possible to obtain maximal cell viability15.</li>
	<li>Centrifuge the PBMCs at 400 x g for 10 min.</li>
	<li>Resuspend the pellet with 5 ml RPMI 1640 complete medium and count the cells.</li>
	<li>Measure cell viability by established methods such as trypan blue exclusion method. Generally cell viability with this method is about 90 - 95%. The PBMCs are now ready to use in experiments.</li>
</ol>
4. Cell Death Determination

NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be changed accordingly.
<ol>
	<li>Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well, as indicated below.</li>
	<li>Dilute WS-CM to the following concentrations: 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, and 5 &#181;g/mL of equi-nicotine units (based on the nicotine content in WS-CM)12.</li>
	<li>Dilute nicotine in RPMI medium to the following concentrations: 100, 200, 500, 750, 1000, 2000, and 3000 &#181;g/ml. CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs suspended in RPMI complete medium to each well at a concentration of 1 x 106 cells/well. The total volume of cells plus WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>For the purpose of this study, prepare two sets of plates as above. Cover the plates and incubate one plate for 24 hr and one plate for 3 hr at 37 &#176;C and 5% CO2. Adjust time points as necessary.</li>
	<li>Wash the cells at RT by centrifuging at 300 x g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally resuspending cells with 200 &#181;l of ice cold running buffer, and repeat the washing step one more time.</li>
	<li>Add 95 &#181;l of running buffer followed by 5 &#181;l of 7-aminoactinomycin D (7AAD) to each well for a total volume of 100 &#181;l/well. Incubate the plate in the dark at RT for 15 min.</li>
	<li>Add 100 &#181;l of running buffer to cluster tubes. Transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on flow cytometer.</li>
	<li>Determine the percentage of 7AAD-positive cells using flow cytometry analysis software.</li>
</ol>
5. EC50 Determination
<ol>
	<li>The EC50 values of WS-CM and nicotine are determined by 7AAD-positive staining of PBMCs.</li>
	<li>The EC50 value is defined as the concentration at which 50% of the cells were no longer viable in a 24 hr assay, and the values are expressed as &#956;g of equi-nicotine units/ml.</li>
	<li>For the purposes of this study, the EC50 values were determined to be 1.56 &#956;g/ml and 1,650 &#181;g/ml for WS-CM and nicotine, respectively.</li>
</ol>
6. Secreted Cytokines
NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be adjusted accordingly.
<ol>
	<li>Dilute WS-CM in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well at the concentration of 0.3, 1.56, 3, and 5 &#181;g/ml of equi-nicotine units.</li>
	<li>Dilute nicotine to the following concentrations: 100, 200, 500, 750, 1000, 2000, and 3000 &#181;g/mL. CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs suspended in RPMI complete medium to each well at a concentration of 1 x 106 cells/well. The total volume of cells plus WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>Cover the plate and incubate for 3 h at 37 &#176;C and 5% CO2.</li>
	<li>Wash the cells at RT by centrifuging at 300 x g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally suspending cells with 200 &#181;l of ice cold running buffer and repeating the washing step one more time.</li>
	<li>Add 200 &#181;l of RPMI complete medium, and repeat the washing step one more time.</li>
	<li>Add 200 &#181;l of 10 &#181;g/ml LPS medium to each well.</li>
	<li>Cover the plate and incubate for 4 hr, 24 hr, 48 hr, or 72 hr at 37 &#176;C and 5% CO2. Adjust incubation times as necessary.</li>
	<li>Centrifuge the plate at 300 &#215; g for 3 min.</li>
	<li>Take 175 &#181;l of supernatant from each well and store in a freezer at -80 &#176;C to perform the assays in steps 7 and 8.</li>
</ol>
7. Cytometric Bead Array Assay
<ol>
	<li>Thaw the cell supernatants prepared from step 6.10 and use in the CBA assay. Perform the cytometric bead array (CBA) assay as per the manufacturer&#8217;s instructions.</li>
</ol>
8. IL-8 ELISA
<ol>
	<li>Thaw the cell supernatants from step 6.10 and use in the IL-8 ELISA. Perform the ELISA assay as per the manufacturer&#8217;s instructions.</li>
</ol>
9. Intracellular Staining and Flow Cytometry
NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be adjusted accordingly.
<ol>
	<li>Dilute WS-CM in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well at the concentration of 0.3, 1.56, 3, and 5 &#181;g/ml of equi-nicotine units.</li>
	<li>In the same plate, dilute nicotine to the following concentrations: 2, 10, 50, 100, 500, 2000, and 4000 &#181;g/ml. CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs suspended in RPMI complete medium at a concentration of 1 &#215; 106 cells/well. The total volume of cells plus WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>Cover the plate and incubate for 3 hr at 37 &#176;C and 5% CO2.</li>
	<li>Wash the cells at RT by centrifuging at 300 &#215; g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally resuspending cells with 200 &#181;l of ice cold running buffer, and repeat the washing step one more time.</li>
	<li>Add 200 &#181;l of RPMI complete medium to the plate, and repeat the wash step one more time.</li>
	<li>Prepare the working concentrations of 2 &#181;l/ml GolgiPlug and 10 &#181;g/ml LPS using RPMI complete medium and add 200 &#181;l to each well.</li>
	<li>Incubate the plate for 6 hr at 37 &#176;C and 5% CO2.</li>
	<li>At the end of step 9.8, wash the cells with running buffer (4 &#176;C) and spinning at 300 x g for 3 min at 4 &#176;C.</li>
	<li>Add 100 &#181;l of Cytofix to each well and incubate for 20 min at 4 &#176;C.</li>
	<li>Wash the cells 3 times as described in step 9.9 with 1x Permwash (4 &#176;C) at 300 x g for 3 min at 4 &#176;C.</li>
	<li>Add 45 &#181;L of 1x Cytoperm to each well followed by 5 &#181;l of each of one of the following antibodies to each well: TNF-&#945;-Alexa Fluor 488, IFN-&#947; V500, MIP-1&#945; PE. Incubate at 4 &#176;C for 30 min.</li>
	<li>Wash the cells two times as described in step 9.9 with 1x Permwash (4 &#176;C) and one time with running buffer (4 &#176;C) at 300 x g for 3 min at 4 &#176;C.</li>
	<li>Resuspend the cells with 200 &#181;l of 2% paraformaldehyde (4 &#176;C). Transfer the cells into 12 &#215; 75 mm tubes, and analyze the samples on flow cytometer. CAUTION: Paraformaldehyde is corrosive, acutely toxic and a health hazard.</li>
</ol>
10. K562 Killing Assay
NOTE: K562 cells should be grown in culture at 37 &#176;C and 5% CO2 with RPMI complete medium until they reach 80% confluence before the assay.
<ol>
	<li>Prepare a 5 mM carboxyfluorescein succinimidyl ester (CFSE) stock solution by adding 18 &#181;l of DMSO to the vial.</li>
	<li>Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well to achieve the desired equi-nicotine units or nicotine concentrations for each well. CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs into RPMI complete medium at a concentration of 1.5 x 106 cells/well. The total volume of cells with WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>Cover the plate and incubate for 3 hr at 37 &#176;C and 5% CO2.</li>
	<li>Wash the K562 cells by adding 10 ml of DPBS and centrifuge at 400 x g for 8 min at RT.</li>
	<li>Resuspend the cells with 10 ml of DPBS and count the K562 cells.</li>
	<li>Prepare CFSE working solution by adding 1 &#181;l of CFSE stock solution to 1 ml DPBS.</li>
	<li>Add 1 ml of the CFSE working solution to 1 ml of the K562 cell suspension containing 1 - 2 x 107 cells. Vortex and incubate precisely for 2 min at RT.</li>
	<li>Immediately add 200 &#956;l of FBS. Vortex and incubate precisely for 2 min at RT.</li>
	<li>Add 10 ml of RPMI complete medium and centrifuge the tube at 400 x g for 8 min at RT.</li>
	<li>Remove the RPMI supernatant and break the pellet and resuspend the cells with 10 ml of RPMI complete medium and count the CFSE-labeled K562 cells.</li>
	<li>Wash the PBMCs by centrifuging the plate at 300 x g for 3 min at RT. Aspirate the supernatant by decanting the liquid. Replace the cover and vortex the bottom of the plate. Add 200 &#181;l of RPMI complete medium and repeat washing step one more time.</li>
	<li>Add CFSE-labeled K562 cells at a ratio of 1:15 (100,000 K562s:1.5 x 106 PBMCs) to each sample well of the PBMC plate and further incubate for 5 hr at 37 &#176;C and 5% CO2.</li>
	<li>Wash the cell mix by centrifuging at 300 x g for 3 min at RT. Aspirate the supernatant by discarding the liquid. Place the cover and vortex the bottom of the plate.</li>
	<li>Add 200 &#181;l of running buffer and repeat the washing step described in step 10.14 one more time.</li>
	<li>Add 95 &#181;l of running buffer followed by 5 &#181;l of 7AAD to each well for a total volume of 100 &#181;l/well. Incubate the plate in the dark at RT for 15 min.</li>
	<li>Add 100 &#181;l of running buffer to each well and transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on flow cytometer.</li>
	<li>Determine the percentage of 7AAD-positive and CFSE-positive cells using flow cytometry analysis software.</li>
</ol>

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The authors declare that there are no conflicts of interest.

We and others have previously demonstrated that treatment of PBMCs with TPPs suppresses several responses, including expression and secretion of cytokines and functional measures such as target cell killing14. The experimental methods described in the previous work require longer incubation periods and were modest in magnitude14. Given the potential applications of this attractive ex vivo model for basic and applied research, we investigated whether any of the assay parameters in these mult...

A substantial body of knowledge points to the adverse health effects of chronic cigarette smoking, including cardiovascular disease (CVD), chronic obstructive pulmonary disease (COPD) and cancer1,2. Chronic cigarette smoking has been known to cause inflammation and immune suppression, and these alterations are reported to contribute to increased risk of microbial infection and cancer in smokers3. In vitro and ex vivo techniques are useful in elucidating the molecular basis of the pathophysiological effects of cigarette smoke4-9 (Table 1) and are recognized as important tools for guiding the emerging regulation of various tobacco products10,11.
For example, we have demonstrated that combustible tobacco product preparations (TPPs) such as whole smoke-conditioned medium (WS-CM) and total particulate matter (TPM) are far more cytotoxic and damaging to DNA than non-combustible TPPs or nicotine12,13. Consistent with the published work, it was recently reported that combustible TPPs or nicotine12,13. Consistent with the published work, we recently reported that combustible TPPs caused marked immunosuppression. This was evidenced by suppression of Toll- like receptor (TLR)-ligands, stimulated cytokine secretion, and targeted cell (K562) killing by PBMCs in an ex vivo model14. Given the significance of inflammation in cigarette smoke-induced disease processes, further optimization of the assay conditions to evaluate the immune modulatory effects of cigarette smoke is presented in this report.
The ex vivo assays typically measured intracellular and secreted cytokines as well as the cytolytic function of cytotoxic T and NK cells in K562 cell killing assays14. The assays involved pre-incubation with WS-CM and nicotine and subsequent stimulation of PBMCs with TLR agonists over a period of 3 days; the final readouts are performed using enzyme-linked immunosorbent assays (ELISAs) and/or flow cytometry. We utilized bacterial lipopolysaccharide (LPS), which binds to TLR-4 receptors and stimulates PBMCs resulting in the production of intracellular cytokines and secretion of cytokines. In addition to optimization of the various assay steps for evaluating the immunomodulatory effects of TPPs, we also present methods for isolating PBMCs, cell death assays, and IL-8 quantification. These methods may be applied to address other research questions and further refined to evaluate tobacco products in the regulatory context.
Table 1. Published reports of in vitro and ex vivo methods used to study varilus pathophysiological effects of tobacco product preparations. CS, cigarette smoke medium; CSC, cigarette smoke condensate; CSE, cigarette smoke extract; ELISA, enzyme-linked immunosorbent assay; GADPH, glyceraldehyde 3-phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction; RT, real time quantitative polymerase chain reaction; TS, tobacco smoke.
<table>
	<tbody>
		<tr>
			<td>Author (Year of study)</td>
			<td>Laan et al. (2004)</td>
			<td>Moodie et al. (2004)</td>
			<td>Oltmanns et al. (2005)</td>
			<td>Vayssier (1998)</td>
			<td>Witherden et al. (2004)</td>
			<td>Birrell et al. (2008)</td>
		</tr>
		<tr>
			<td>Cells used</td>
			<td>Human bronchial endothelium cells (BEAS-2B), human neutrophils</td>
			<td>Human alveolar epithelial cells (A549)</td>
			<td>Human airway smooth muscle cells (HASMC)</td>
			<td>Human premonocytic U937 cells, human monocytes</td>
			<td>Alveolar type II epithelial cells (ATII)</td>
			<td>Human monocytic cell line (THP-1), human lung macrophages</td>
		</tr>
		<tr>
			<td>TPP used</td>
			<td>CSE</td>
			<td>CSC</td>
			<td>CSE</td>
			<td>TS</td>
			<td>CSE</td>
			<td>CS</td>
		</tr>
		<tr>
			<td>Method used</td>
			<td>ELISA, qPCR, migration, electromobility shift</td>
			<td>Immunohisto-chemistry, electrophoresis, Arrayscan kit, RT-PCR, ELISA</td>
			<td>ELISA, RT-PCR, qPCR, electrophoresis</td>
			<td>Gel-mobility shift</td>
			<td>Light microscopy, electron microscopy, electrophoresis, ELISA</td>
			<td>qPCR, ELISA, E-toxate kit (Sigma), p65 plate assay (TransAM), electrophoresis, various immunoassay kits</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>IL-8, GM-CF, AP-1, NF-&#954;B, migration</td>
			<td>Histone acetyltransferases, histone deacetylases, NF-&#954;B, IL-8, p-I &#954;B-&#945;, GADPH</td>
			<td>HO-1, GADPH, RANTES, IL-8, eotaxin</td>
			<td>Heat shock/stress proteins (HSP/Hsp70), HF transcription factor, NF-&#954;B, TNF-&#945;</td>
			<td>Surfactant protein (SP-A, SP-C), IL-8, MCP-1, GRO-&#945;, TNF-&#945;, IL-1&#946;, IFN-&#947;</td>
			<td>IL-8, IL-1&#946;, IL-6, TNF-&#945;, MIP1-&#945;, GRO-&#945;, MAPK/JNK/ERK phosphorylation, cJUN:DNA binding, glutathione, p65:DNA binding</td>
		</tr>
		<tr>
			<td>End result</td>
			<td>CSE down-regulates cytokine production via suppression of AP-1 activation.</td>
			<td>H2O2 and CSC enhance acetylation of histone proteins, decrease histone deacetylase activity, differentially regulate proinflammatory cytokine release.</td>
			<td>Cigarette smoke may cause the release of IL-8 from HASMC, enhanced by TNF-&#945;, 20% CSE less IL-8 release, Inhibition of eotaxin and RANTES by cigarette smoke.</td>
			<td>TS activated HF transcription factor, which was associated with Hsp70 overexpression and inhibition of NFkB binding activity and TNF-&#945; release.</td>
			<td>Reduced ATII cell-derived chemokine levels compromise alveolar repair, contributing to cigarette smoke-induced alveolar damage and emphysema.</td>
			<td>Data provide mechanistic explanation for why smokers have increased respiratory infections. Suppression of the innate response is accompanied by an increase in IL-8.</td>
		</tr>
	</tbody>
</table>

<table><tbody><tr><td>12 x 75 mm tubes</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>2 ml microtubes</td><td>Axygen</td><td>MCT-150-C-S</td><td></td></tr><tr><td>3R4F reference cigarettes</td><td>Univ. of Kentucky, College of Agriculture</td><td>3R4F</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>500 ml bottle</td><td>Corning</td><td>430282</td><td></td></tr><tr><td>7AAD</td><td>BD Pharmingen</td><td>559925</td><td></td></tr><tr><td>96-well flat bottom plate</td><td>Termo Nunc</td><td>439454</td><td></td></tr><tr><td>96-well round bottom plates</td><td>BD Falcon</td><td>353077</td><td></td></tr><tr><td>Cell culture hood</td><td>Thermo Scientific</td><td>1300 Series A2</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>58110R</td><td></td></tr><tr><td>CFSE</td><td>Molecular Probes Life Technologies</td><td>C34554</td><td></td></tr><tr><td>Cluster tubes</td><td>Corning</td><td>4401</td><td>Harmful if swallowed, carcinogen</td></tr><tr><td>Cytofix/Cytoperm (Permwash)</td><td>BD Biosciences</td><td>555028</td><td>Flammable</td></tr><tr><td>DMSO (dimethyl sulfoxide )</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>DPBS</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>FCAP Array</td><td>BD Biosciences</td><td>652099</td><td>Software analyzes CBA data</td></tr><tr><td>Filter unit</td><td>Nalgene</td><td>156-4020</td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td>FACS Canto II</td><td>8 colors, at Ex 405 and Em 785.</td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td>FACS Calibur</td><td>4 colors at Ex 495 and Em 785.</td></tr><tr><td>Flow cytometry analysis software</td><td>Tree Star</td><td>FlowJo</td><td></td></tr><tr><td>Freezing container</td><td>Nalgene</td><td>5100-0001</td><td>Contains DMSO, irritant</td></tr><tr><td>GogliPlug</td><td>BD Biosciences</td><td>555029</td><td>Carcinogen, irritant, corrosive</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>339741</td><td></td></tr><tr><td>Human Inflammatory Cytokine Kit</td><td>BD Biosciences</td><td>551811</td><td></td></tr><tr><td>IFN-&amp;gamma; V-500 Antibody</td><td>BD Horizon</td><td>561980</td><td>skin sensitizer</td></tr><tr><td>IL-8 ELISA Kit</td><td>R and D Systems</td><td>DY208</td><td></td></tr><tr><td>Isolation buffer</td><td>Isolymph, CTL Scientific Corp.</td><td>1114868</td><td>Flammable liquid, irritant</td></tr><tr><td>Isopropyl alcohol</td><td>Sigma-Aldrich</td><td>W292907</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>LPS</td><td>Sigma-Aldrich</td><td>L2630</td><td></td></tr><tr><td>MIP1-&amp;alpha; PE Antibody</td><td>BD Pharmingen</td><td>554730</td><td>Acute toxicity, oral</td></tr><tr><td>Monensin</td><td>Sigma-Aldrich</td><td>M5273</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Nicotine</td><td>Sigma-Aldrich</td><td>N3876</td><td>Acute toxicity, environmental hazard</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>&amp;ldquo;M&amp;rdquo;</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Flammable, skin irritation</td></tr><tr><td>Pen/strep</td><td>Gibco Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>Running buffer</td><td>MACS Running Buffer, Miltenyi Biotech</td><td>130-091-221</td><td></td></tr><tr><td>Th1/Th2 CBA Kit</td><td>BD Biosciences</td><td>551809</td><td></td></tr><tr><td>TNF-&amp;alpha; Alexa Fluor 488 Antibody</td><td>BioLegend</td><td>502915</td><td></td></tr><tr><td>Transfer pipette</td><td>Fisher Scientific</td><td>13-711-20</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr></tbody></table>

methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

The results were presented as mean &#177; standard error of the mean (four donor samples). The student&#8217;s t-test between treated and untreated control samples was performed using Excel software as well as t-test comparisons for all treatments with their corresponding controls. The statistical significance was indicated by: *, P &#60; 0.05; **, P &#60; 0.005; ***, P &#60; 0.0005.
To measure the effect of exposure to WS-CM and nicotine, PBMCs were treated with different concentrations of WS...

Watch this Scientific Journal Video about Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations at JoVE.com

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

methods-to-evaluate-cytotoxicity-immunosuppression-combustible

Research

JoVE Journal

Immunology and Infection

11.4K Views.  Wake Forest University Health Sciences. Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure. 

Video: Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Medicine

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Bioengineering

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems.

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and ...

Chemistry

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Biology

Analyzing the Effect of Tobacco Product Preparations on Cytokine Production via Intracellular Staining and Flow Cytometry

Source: Arimilli, S. et al., <a href="https://app.jove.com/v/52351">Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates the effect of tobacco product preparations, TPPs, on cytokine production from human peripheral blood mononuclear cells using flow cytometry. Treatment of the cells with TPP downregulates the production of pro-inflammatory cytokines, which is estimated after lipopolysaccharide stimulation, followed by immunostaining and flow cytometry analysis.

Source: Arimilli, S. et al., Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

An Assay to Measure Chemically-Induced Cytotoxicity in Human Precision-Cut Lung Slices

Source: Neuhaus, V. et al., <a href="https://app.jove.com/v/57042">Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.</a>&#160;J. Vis. Exp.&#160;(2018)
This video illustrates a technique for evaluating cytotoxicity in human precision-cut lung slices through the WST-1 assay. Human precision-cut lung slices are subjected to incremental concentrations of a cytotoxic chemical to quantify their cytotoxicity. A decrease in color intensity serves as confirmation of the chemical&#39;s detrimental impact on lung cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. General Preparation of Human ...

Immunodiagnostics

In Vitro and In Vivo Models

Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations

Source: Arimilli, S. et al., <a href="https://app.jove.com/v/52351">Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations.</a> J. Vis. Exp. (2015)
In this video, we study the effect of a whole smoke-conditioned medium, a combustible tobacco product preparation, on K562 cell killing by effector peripheral blood mononuclear cells.

Source: Arimilli, S. et al., Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations. J. Vis. Exp. (2015)
In ...

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

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