Name: 방법은 가연성 담배 제품 준비의 세포 독성 및 면역 억제를 평가하기 (동영상) | JoVE Journal
Uploaded: 2015-01-10T16:00:00.000+00:00
Duration: 9 min 25 s
Description: 학습하세요 인간 말초혈액 단핵세포(PBMC)를 이용한 가연성 담배 제품의 세포 독성 및 면역 억제 평가 방법을. 이 기법은 담배 노출이 면역 기능에 미치는 영향을 연구하는 데 활용됩니다.

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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>

<ol><li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22How+Tobacco+Smoke+Causes+Disease%3A+The+Biology+and+Behavioral+Basis+for+Smoking-Attributable+Disease%2C+A+Report+of+the+Surgeon+General%22">How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease, A Report of the Surgeon General</a>. , U.S. Department of Health and Human Services. (2010).</li>
<li>Zeller, M., Hatsukami, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Strategic+Dialogue+on+Tobacco+Harm+Reduction%3A+a+vision+and+blueprint+for+action+in+the+US%5BTitle%5D)+AND+%22Tob.+Control%22%5BJournal%5D)">The Strategic Dialogue on Tobacco Harm Reduction: a vision and blueprint for action in the US.</a> Tob. Control. 18, 324-332 (2009).</li>
<li>Sopori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+cigarette+smoke+on+the+immune+system%5BTitle%5D)+AND+%22Nature+Reviews+Immunology%22%5BJournal%5D)">Effects of cigarette smoke on the immune system.</a> Nature Reviews Immunology. 2, 372-377 (2002).</li>
<li>Birrell, M. A., Wong, S., Catley, M. C., Belvisi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+tobacco-smoke+on+key+signaling+pathways+in+the+innate+immune+response+in+lung+macrophages%5BTitle%5D)+AND+%22J.+Cell+Physiol%22%5BJournal%5D)">Impact of tobacco-smoke on key signaling pathways in the innate immune response in lung macrophages.</a> J. Cell Physiol. 214, 27-37 (2008).</li>
<li>Laan, M., Bozinovski, S., Anderson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cigarette+smoke+inhibits+lipopolysaccharide-induced+production+of+inflammatory+cytokines+by+suppressing+the+activation+of+activator+protein-1+in+bronchial+epithelial+cells%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Cigarette smoke inhibits lipopolysaccharide-induced production of inflammatory cytokines by suppressing the activation of activator protein-1 in bronchial epithelial cells.</a> J. Immunol. 173, 4164-4170 (2004).</li>
<li>Moodie, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidative+stress+and+cigarette+smoke+alter+chromatin+remodeling+but+differentially+regulate+NF-kappaB+activation+and+proinflammatory+cytokine+release+in+alveolar+epithelial+cells%5BTitle%5D)+AND+%22Faseb+J%22%5BJournal%5D)">Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinflammatory cytokine release in alveolar epithelial cells.</a> Faseb J. 18, 1897-1899 (2004).</li>
<li>Oltmanns, U., Chung, K. F., Walters, M., John, M., Mitchell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cigarette+smoke+induces+IL-8%2C+but+inhibits+eotaxin+and+RANTES+release+from+airway+smooth+muscle%5BTitle%5D)+AND+%22Respir.+Res%22%5BJournal%5D)">Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle.</a> Respir. Res. 6, 74(2005).</li>
<li>Vayssier, M., Favatier, F., Pinot, F., Bachelet, M., Polla, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco+smoke+induces+coordinate+activation+of+HSF+and+inhibition+of+NFkappaB+in+human+monocytes%3A+effects+on+TNFalpha+release%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">Tobacco smoke induces coordinate activation of HSF and inhibition of NFkappaB in human monocytes: effects on TNFalpha release.</a> Biochem. Biophys. Res. Commun. 252, 249-256 (1998).</li>
<li>Witherden, I. R., Vanden Bon, E. J., Goldstraw, P., Ratcliffe, C., Pastorino, U., Tetley, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+human+alveolar+type+II+epithelial+cell+chemokine+release%3A+effects+of+cigarette+smoke+and+neutrophil+elastase%5BTitle%5D)+AND+%22Am.+J.+Respir.+Cell+Mol.+Biol%22%5BJournal%5D)">Primary human alveolar type II epithelial cell chemokine release: effects of cigarette smoke and neutrophil elastase.</a> Am. J. Respir. Cell Mol. Biol. 30, 500-509 (2004).</li>
<li>Hatsukami, D. K., Biener, L., Leischow, S. J., Zeller, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco+and+nicotine+product+testing%5BTitle%5D)+AND+%22Nicotine+Tob.+Res%22%5BJournal%5D)">Tobacco and nicotine product testing.</a> Nicotine Tob. Res. 14, 7-17 (2012).</li>
<li>Ashley, D. L., Backinger, C. L., van Bemmel, D. M., Neveleff, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco+Regulatory+Science%3A+Research+to+Inform+Regulatory+Action+at+the+Food+and+Drug+Administration%27s+Center+for+Tobacco+Products%5BTitle%5D)+AND+%22Nicotine+Tob.+Res%22%5BJournal%5D)">Tobacco Regulatory Science: Research to Inform Regulatory Action at the Food and Drug Administration's Center for Tobacco Products.</a> Nicotine Tob. Res. , (2014).</li>
<li>Arimilli, S., Damratoski, B. E., Bombick, B., Borgerding, M. F., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+cytotoxicity+of+different+tobacco+product+preparations%5BTitle%5D)+AND+%22Regul.+Toxicol.+Pharmacol%22%5BJournal%5D)">Evaluation of cytotoxicity of different tobacco product preparations.</a> Regul. Toxicol. Pharmacol. 64, 350-360 (2012).</li>
<li>Gao, H., Prasad, G. L., Zacharias, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+cell-specific+cytotoxic+responses+of+oral+cavity+cells+to+tobacco+preparations%5BTitle%5D)+AND+%22Toxicol+In+Vitro%22%5BJournal%5D)">Differential cell-specific cytotoxic responses of oral cavity cells to tobacco preparations.</a> Toxicol In Vitro. , (2012).</li>
<li>Arimilli, S., Damratoski, B. E., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combustible+and+non-combustible+tobacco+product+preparations+differentially+regulate+human+peripheral+blood+mononuclear+cell+functions%5BTitle%5D)+AND+%22Toxicol.+In+Vitro%22%5BJournal%5D)">Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions.</a> Toxicol. In Vitro. 27, 1992-2004 (2013).</li>
<li>Arimilli, S., Damratoski, B. E., Chen, P., Jones, B. A., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+isolation+of+leukocyte+subsets+from+fresh+and+cryopreserved+peripheral+blood+mononuclear+cells+in+clinical+research%5BTitle%5D)+AND+%22Cryo+Letters%22%5BJournal%5D)">Rapid isolation of leukocyte subsets from fresh and cryopreserved peripheral blood mononuclear cells in clinical research.</a> Cryo Letters. 33, 376-384 (2012).</li>
<li>Schmid, I., Krall, W. J., Uittenbogaart, C. H., Braun, J., Giorgi, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dead+cell+discrimination+with+7-amino-actinomycin+D+in+combination+with+dual+color+immunofluorescence+in+single+laser+flow+cytometry%5BTitle%5D)+AND+%22Cytometry%22%5BJournal%5D)">Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.</a> Cytometry. 13, 204-208 (1992).</li>
<li>Kim, G. G., Donnenberg, V. S., Donnenberg, A. D., Gooding, W., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+multiparametric+flow+cytometry-based+cytotoxicity+assay+simultaneously+immunophenotypes+effector+cells%3A+comparisons+to+a+4+h+51Cr-release+assay%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.</a> J. Immunol. Methods. 325, 51-66 (2007).</li>
<li>Taga, K., Yamauchi, A., Kabashima, K., Bloom, E. T., Muller, J., Tosato, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Target-induced+death+by+apoptosis+in+human+lymphokine-activated+natural+killer+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Target-induced death by apoptosis in human lymphokine-activated natural killer cells.</a> Blood. 87, 2411-2418 (1996).</li>
</ol>

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

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 ± standard error of the mean (four donor samples). The student’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 &lt; 0.05; **, P &lt; 0.005; ***, P &lt; 0.0005.
To measure the effect of exposure to WS-CM and nicotine, PBMCs were treated with different concentrations of WS-CM and nicoti...

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 ...

The overall goal of this procedure is to evaluate the effects of tobacco product preparation on cytokine secretion and target cell killing by P BMCs. This is accomplished by first treating cultured P BMCs with whole smoke condition, medium or nicotine, and then intracellular staining of the treated p BMCs is performed to determine their cytokine production. Alternatively, CFSE labeled K 5 62 cells are co-culture with whole smoke conditioned medium treated PBMC effector cells and the viability of the K 5 62 cells is qualified by seven A a d staining.Ultimately, the effects of the combustible tobacco product preparations on the P BMCs from either assay are measured by flow cytometry. The main advantages of our techniques or the existing methods, such as chromium release assay or ALI spot are our methods are high throughput, non-radioactive, and we can measure the cell functionality. Though this method can provide insight into the biological effects of tobacco product preparations.It can also be applied to the evaluation of other compounds that impact the tolike receptor mediated and STIC functions of PBMC effector cells Before beginning the staining procedure. Dilute whole smoke cond

methods-to-evaluate-cytotoxicity-immunosuppression-combustible

Research

연구

JoVE Journal

Immunology and Infection

면역학 및 감염학

방법은 가연성 담배 제품 준비의 세포 독성 및 면역 억제를 평가하기

폐 상피 세포는 생쥐의 호흡기로부터 격리하고 차별 호흡기 상피의 모델로 공기 - 액체 인터페이스에서 교양 수 있습니다. 프로토콜이 환경 독소에 분자 반응을 연구하기 위해, culturing를 분리하고 주류 담배 연기 이러한 세포를 노출에 대해 설명합니다.

에어 리퀴드 인터페이스에서 마우스 호흡기 상피 세포 및 실험 담배 연기에 노출의 분리

이 비디오는 유세포 분석을 사용하여 담배 제품 제제(TPP)가 인간 말초 혈액 단핵 세포의 사이토카인 생산에 미치는 영향을 보여줍니다. TPP로 세포를 처리하면 전염증성 사이토카인의 생성을 하향 조절하며, 이는 지질다당류 자극 후 추정된 후 면역염색 및 유세포 분석 분석이 수행됩니다.

세포 내 염색 및 유세포 분석을 통한 사이토카인 생산에 대한 담배 제품 제제의 효과 분석

이 비디오에서는 가연성 담배 제품 제제인 전체 연기 조절 매체가 효과기 말초 혈액 단핵 세포에 의한 K562 세포 사멸에 미치는 영향을 연구합니다.

담배 제품 제제에 노출된 말초 혈액 단핵 세포에 의한 K562 세포 사멸

이 비디오는 WST-1 분석을 통해 인간의 정밀 절단 폐 절편에서 세포 독성을 평가하는 기술을 보여줍니다. 인간의 정밀하게 절단된 폐 절편은 세포 독성을 정량화하기 위해 세포 독성 화학 물질의 증분 농도에 노출됩니다. 색상 강도의 감소는 화학 물질이 폐 세포에 미치는 해로운 영향을 확인하는 역할을 합니다.

인간의 정밀 절단 폐 절편에서 화학적으로 유도된 세포 독성을 측정하기 위한 분석

이 프로토콜의 목적은 인체 유기형 3D 기관지 및 코 조직 모델을 공기-액체 계면에서 주류 담배 연기(CS)에 노출시키는 것입니다. 그런 다음 시토크롬 P450 활성 분석, 섬모 박동 측정 및 시스템 생물학 접근 방식을 사용하여 CS가 조직에 미치는 영향을 조사합니다.

전체 담배 연기에의 Organotypic 3D 기관지와 코 조직 문화 모델의 반복 노출의 영향 평가

이 연구의 목적은 임피던스 기반 실시간 세포 분석기와 체외 독성 평가를 위한 HCS(High-Content Screening) 기반 플랫폼의 조합을 사용하여 15가지 담배 연기 성분의 생물학적 영향을 평가하는 것이었습니다. 이 연구는 테스트된 화합물의 효과적인 용량, 독성 및 작용 모드에 대한 정보를 제공합니다.

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

이 프로토콜은 설치류를 전자 담배 (E-vapor) 및 담배 연기에 노출시키는 방법을 설명합니다. 노출 챔버는 설치류에 전자 증기 또는 담배 연기를 전달하는 자동화 된 펌핑 시스템으로 마취 챔버를 개조하여 구성됩니다. 이 시스템은 많은 실험적 엔드 포인트를 수용하도록 쉽게 수정할 수 있습니다.

전자 담배와 담배 연기의 효과 비교<em&gt; In Vivo</em&gt; 노출 시스템

여기, 우리는 기존의 담배 연기 및 전자 담배에 어로 졸의 추출 물을 만드는 데 사용할 수 있는 프로그래밍 가능한 실험실 장치를 설명 합니다. 이 메서드는 일반 담배와 전자 담배 사이의 직접 비교를 하기 위한 유용한 도구를 제공 하며 전자 담배 연구를 액세스할 수 있는 진입점입니다.

전통적인 담배 연기와 전자 담배 연 무질에서 액체 추출의 세대에 대 한 장치를 운영 하는 마이크로컨트롤러

3Rs 원리에 비추어 호흡기 모델 동물 연구 대안으로 진화 됩니다. 특히 호흡 물질의 위험 평가 대 한 적절 한 분석의 부족이 이다. 여기, 우리는 공 수 물질의 평가 대 한 인간의 정밀 컷 폐 슬라이스를 사용 하 여를 설명합니다.

세포 독성의 평가 인간의 정밀 컷 폐 분할 영역에서 물질의 Immunomodulatory 효과

전자 담배 (전자-cig) 사용자는 전 세계적으로 증가 하 고 있다. 그러나 작은,, 전자 cig 흡입된에 어로 졸에 의해 유도 된 건강 효과 대 한 알려져 있다. 이 문서는 전자 cig에 어로 졸 생성 기법 동물 노출 및 후속 독물학 연구를 위한 적합 한 설명. 이러한 프로토콜 실험적으로 재현 하 고 표준화 된 전자 cig 노출 시스템을 설정 해야 합니다.

3 세대 기계 Vaping 장치에서 전자 담배에 어로 졸의 생성: 독물학 연구 응용 프로그램

우리는 공기-액체 계면에서 대마초 증기 노출을 연구하기 위한 표준화된 체외 모델을 수립합니다. 이 모델은 기화된 대마초의 영향을 조사하기 위한 체계적인 접근 방식을 제공하여 흡연의 대안으로 대마초 기화기에 대한 증가하는 관심을 해결합니다.

시험관 내 공기-액체 계면에서 폐 상피 세포의 대마초 노출

학습하세요 인간 말초혈액 단핵세포(PBMC)를 이용한 가연성 담배 제품의 세포 독성 및 면역 억제 평가 방법을. 이 기법은 담배 노출이 면역 기능에 미치는 영향을 연구하는 데 활용됩니다.

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

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