这项工作是由雷诺烟草公司（RJRT）根据与维克森林大学医学院的合作研究协议投资。 GL Prasad是RJRT的全职员工。

注:书面知情同意做这项研究在当地的临床研究单位根据IRB批准获得，每良好的临床实践。血液处理中，在无菌条件下执行PBMC和其它细胞培养实验的隔离，使用微生物的无菌用品和试剂。 1，WS-CM准备<ol><li>产生的WS-CM如前所述12。 <ol><li>准备WS-CM由四个3R4F参考香烟通过罗斯威尔公园纪念研究所（RPMI）1640培养基无酚红用下面的吸烟方案通过烟:35-60-2，在毫升抽吸量，以秒为单位的时间间隔粉扑，粉扑和持续时间以秒为单位，分别为。每种制剂产生20ml的样品。 </li></ol></li><li>标签管（S）与日期，时间完成，香烟名称和编号。存储500微升等分在-80℃吸烟后立即完成。 </li><li>分析冷冻WS等分-CM确定尼古丁，烟草特异性亚硝胺，和多环芳香烃的含量如前所述12。 </li></ol> 2.分离外周血单个核细胞<ol><li>收集来自健康捐赠者（谁是非消费者对烟草制品）新鲜血液隔离从新鲜血液的外周血单个核细胞在维克森林浸礼会教友健康IRB 15一个独立的审批如下所述。 <ol><li>在此之前的血袋的到来，具有的500毫升瓶中，剪刀，隔离缓冲器，和Dulbecco氏磷酸盐缓冲盐水（DPBS）（RT）准备在生物安全级别2（BSL-2）的细胞培养罩。隔离缓冲器必须避光。 </li><li>握住血袋颠倒并削减略低于它已被夹住造成至少3厘米管的管中。 </li><li>取下一瓶500毫升的上限，按住瓶口上面管。拿起血袋并倒转，使血液从包成吨自由流动他直到瓶的袋子是空的。允许血液流到瓶与直下作为的内壁以避免产生气泡。 </li><li>倾隔离缓冲器入瓶以1:5的比例的隔离缓冲器来的血液。瓶盖瓶口紧紧地，轻轻地颠倒了，最终在年底，10倍。离开瓶在细胞培养罩孵育与灯关闭，1小时，在室温。 </li><li>光，稻草色层将上述血液中积聚。用25毫升血清吸管入50ml锥形管中取出这一层。所收集的量可以从50变化 - 300毫升，这取决于谁捐献的血液的主题。 </li><li>离心管中，在200×g离心在室温10分钟。 </li><li>吸出半透明的上清液，留下暗血色的沉淀。小球会松动，但粘性。 </li><li>吸取3毫升隔离缓冲分成15毫升锥形管。 </li><li>到重新悬浮颗粒，每50毫升浅黄色液体收集的Add20毫升的DPBSTEP 2.1.5。涡旋调匀，并整合来自多个管的悬浮液。该液体含有悬浮的血细胞。 </li><li>用移液管，在步骤2.1.8转移将5ml悬浮的血细胞置于3mL的隔离缓冲器。倾斜的15毫升锥形管，其中包含的细胞悬浮液缓慢并轻轻以创建两个单独的层。离心管中，在400 XG 40分钟在室温以最小的加速和不带刹车。 </li><li>使用移液管，以除去含有的PBMC所得到的混浊的中间层（血沉棕黄层）到50毫升锥形管中。避免绘图等层次分明下方。转移不超过25 ml接种入各50ml锥形管中。 </li><li>加25毫升冷的运行缓冲液（或多种填充50ml锥形管的整个剩余体积）洗细胞。离心细胞，在400×g离心在4℃下10分钟。 </li><li>重悬沉淀用运行缓冲液10毫升这包含外周血单个核细胞。计数细胞，并使用即时通讯的治疗中，或置于冰上冻结。 </li></ol></li></ol> 3.冷冻和解冻的外周血单个核细胞<ol><li>离心收集在步骤2.1.13，对于以400×g的10分钟的PBMC。 </li><li>填充冷冻集装箱，每个制造商的说明异丙醇。警告:异丙醇是易燃，剧毒。 </li><li>重悬的RPMI 1640培养基（4℃）含有20％胎牛血清（FBS）和10％的二甲亚砜（DMSO）中的沉淀。这是RPMI 1640冷冻介质。悬浮颗粒与冷冻介质，这将导致在具有约5×10 7个细胞/ ml的悬浮液的量。可用于冷冻细胞的数量将有所不同。 </li><li>分配1ml等分细胞悬浮于2ml冷冻管并放置冻存管在冷冻容器中。将冷冻容器冷冻管在冷冻在-80°C至存储O / N，然后取出冻存管，并转移到存储在-150℃的低温冷冻至-190℃。 </li><li>除去从低温储藏的冻存管，并在37℃下温和搅拌迅速解冻它在水浴。 </li><li>立即传送解冻的PBMC中冻存管入15ml锥形管中以含有10％FBS，1％青霉素/链霉素和1％L-谷氨酰胺10毫升的RPMI 1640完全培养基（4℃）。解冻冻存管的内容，应尽快转移，以获得最大的细胞活力15。 </li><li>离心的PBMC，在400×g离心10分钟。 </li><li>悬浮用5ml RPMI 1640完全培养基中的小球，并计数细胞。 </li><li>通过已建立的方法，如台盼蓝排除法测定细胞活力。通常用这种方法的细胞生存力为约90 - 95％。在外周血单个核细胞现在准备在实验中使用。 </li></ol> 4.细胞死亡测定注意:这里列出稀释该STU的目的DY。稀释液可以相应地改变。 <ol><li>用RPMI完全培养基至100μl的总体积稀释的WS-CM或尼古丁在一个96孔板/孔，如下所示。 </li><li>稀的WS-CM以下列浓度:0.1，0.5，1,1.5，2，2.5，3，4，和5微克/毫升的相等烟碱单元（基于WS-CM中的尼古丁含量）12。 </li><li>稀尼古丁在RPMI培养基中，以下列浓度:100，200，500，750，1000，2000和3000微克/毫升。注意:尼古丁是急性毒性和对环境有害。 </li><li>加入100微升的PBMC悬浮于RPMI完全培养基中至每个孔1×10 6个细胞/孔的浓度。细胞加的WS-CM或尼古丁的总体积为200微升/孔。 </li><li>本研究的目的，准备两套板如上。覆盖板并孵育一个板放置24小时和一个板3小时，在37℃和5％的CO 2。调整的时间点是必要的。 </li&gt; <li>通过在300×g离心离心3分钟，吸取上清，涡旋板以板覆盖的底部，最后用200微升冰冷的缓冲液重新悬浮细胞洗细胞在RT，并重复该洗涤步骤一次。 </li><li>添加95微升缓冲液，随后用5微升7氨基放线菌素D（7AAD）至每孔100微升的总体积/孔。孵育板在黑暗中在RT下15分钟。 </li><li>加入100微升缓冲液，以集群管。从板到集群管的每个孔中转移细胞悬液的整个体积并采集样品上流式细胞仪。 </li><li>确定使用流式细胞分析软件7AAD阳性细胞的百分比。 </li></ol> 5. EC 50测定<ol><li>对WS-CM和尼古丁的EC 50值由外周血单个核细胞的7AAD阳性染色确定。 </li><li>的EC 50值被定义为浓entration在其中细胞的50％在24小时测定法不再可行，并且这些值被表示为相等尼古丁单位/毫升微克。 </li><li>这项研究的目的，EC 50值确定为1.56微克/毫升和1650微克/毫升的WS-CM和尼古丁，分别。 </li></ol> 6.分泌的细胞因子注意:这里列出的稀释是本研究的目的。稀释液可以相应地调整。 <ol><li>用RPMI完全培养基中加入100μl/孔在的浓度的总体积稀释的WS-CM在一个96孔板0.3，1.56，图3和5微克/毫升的相等尼古丁单元。 </li><li>稀释烟碱至下列浓度:100，200，500，750，1000，2000和3000微克/毫升。注意:尼古丁是急性毒性和对环境有害。 </li><li>加入100微升的PBMC悬浮于RPMI完全培养基中至每个孔1×10 6个细胞/孔的浓度。细胞加的WS-CM或尼古丁的总体积为200微升/孔。 </li><li>覆盖板并孵育在37℃，3小时，5％的CO 2。 </li><li>通过在300×g离心离心3分钟，吸取上清，涡旋板以板覆盖的底部，最后悬浮细胞用运行缓冲液并重复洗涤步骤一次加入200μl冰冷的洗细胞在RT。 </li><li>添加200μl的RPMI完全培养基中，并重复该洗涤步骤一次。 </li><li>加入200μl10微克/毫升的LPS培养基到每个孔中。 </li><li>覆盖板并孵育4小时，24小时，48小时，或72小时，在37℃和5％的CO 2。调整保温时间是必要的。 </li><li>离心板在300×g下3分钟。 </li><li>取175微升上清液从每孔中并储存在冰箱中在-80℃进行测定的步骤7和8。 </li></ol> 7.式微球分析 &lt;OL&gt; <li>解冻步骤6.10和使用在CBA实验制备的细胞上清液。执行流式微球阵列（CBA）测定按照制造商的说明。 </li></ol> 8.的IL-8的ELISA <ol><li>从步骤6.10和使用中的IL-8的ELISA解冻的细胞上清液。执行ELISA测定按照制造商的说明。 </li></ol> 9.细胞内染色和流式细胞仪注意:这里列出的稀释是本研究的目的。稀释液可以相应地调整。 <ol><li>用RPMI完全培养基中加入100μl/孔在的浓度的总体积稀释的WS-CM在一个96孔板0.3，1.56，图3和5微克/毫升的相等尼古丁单元。 </li><li>在相同的板，稀释烟碱至下列浓度:2，10，50，100，500，2000和4000微克/毫升。注意:尼古丁是急性毒性和环境哈萨dous。 </li><li>加入100微升的PBMC悬浮于RPMI完全培养基中以1×10 6个细胞/孔的浓度。细胞加的WS-CM或尼古丁的总体积为200微升/孔。 </li><li>覆盖板孵育3小时，在37℃和5％的CO 2。 </li><li>通过在300×g离心3分钟，吸取上清，涡旋板以板覆盖的底部，最后用200微升冰冷的缓冲液重新悬浮细胞洗细胞在RT，并重复该洗涤步骤一次。 </li><li>添加200μl的RPMI完全培养基中至板，并重复洗涤步骤一次。 </li><li>制备的2微升/毫升GolgiPlug使用RPMI完全培养基中的工作浓度和10微克/毫升的LPS和添加200μl的各孔中。 </li><li>孵育板6小时，在37℃和5％的CO 2。 </li><li>在步骤9.8的端部，用运行缓冲液（4℃）和纺丝，在300洗涤细胞XG为3分钟，在4℃。 </li><li>添加100微升Cytofix的每个孔中，孵育20分钟，在4℃。 </li><li>如在步骤9.9，在300×g离心在4℃下描述，带有1 Permwash（4℃）下进行3分钟洗细胞3次。 </li><li>添加45微升1×Cytoperm向每个孔中，随后用5微升以下每个抗体中的一个，每孔:TNF-α的Alexa Fluor 488，IFN-γ的V500，MIP-1α的PE。孵育在4℃下进行30分钟。 </li><li>如步骤9.9中所述，带有1 Permwash（4℃）和一个时间与运行缓冲液（4℃）在300×g离心3分钟，在4℃下洗涤细胞两次。 </li><li>重悬的细胞用200μl的2％多聚甲醛（4℃）的。转移细胞到12×75毫米管，和血细胞计数分析样品上的流动。注意:多聚甲醛具有腐蚀性，剧毒和健康危害。 </li></ol> 10. K562杀灭试验注:K562细胞应培养在培养于37℃和5％的CO 2的RPMI完全培养基中直到他们达到在测定前80％汇合。 <ol><li>通过加入18微升的DMSO至小瓶制备5mM的羧基琥珀酰亚胺酯（CFSE）原液。 </li><li>用RPMI完全培养基至100μl的总体积/孔以实现所需的等尼古丁单位或尼古丁浓度为每孔稀释的WS-CM或尼古丁在96孔板中。注意:尼古丁是急性毒性和对环境有害。 </li><li>加入100微升的PBMC的进RPMI完全培养基中以1.5×10 6个细胞/孔的浓度。细胞与WS-CM或尼古丁的总体积为200微升/孔。 </li><li>覆盖板孵育3小时，在37℃和5％的CO 2。 </li><li>通过加入10ml的DPBS和离心机在400×g离心在RT 8分钟洗K562细胞。 </li><li>将细胞重悬于10毫升的DPBS和计数的K562细胞。 </li><li>准备CFSE worki纳克溶液中加入1微升的CFSE原液至1ml DPBS。 </li><li>加入1毫升的CFSE工作溶液到1ml含1所述的K562细胞悬浮液- 2×10 7个细胞。涡和孵化正是在室温2分钟。 </li><li>立即加入200μlFBS的。涡和孵化正是在室温2分钟。 </li><li>加入10 mL RPMI完全培养基中，离心反应管，在400×g离心在RT 8分钟。 </li><li>除去上清液的RPMI并打破颗粒和重悬细胞，用10ml RPMI完全培养基中，计数的CFSE标记的K562细胞。 </li><li>通过离心板在300×g离心3分钟，在RT下洗涤的PBMC。通过倾析将液体吸出上清液。重新装上盖子，涡旋盘的底部。添加200μl的RPMI完全培养基中，并重复洗涤步骤一次。 </li><li>添加CFSE标记的K562细胞以1:15的比率:向PBMC板和后续的i的每个样品井（100,000 K562s 1.5×10 6个PBMC中）ncubate 5小时，在37℃和5％的CO 2。 </li><li>通过在300×g离心离心3分钟，在RT下洗涤细胞混合。通过丢弃该液体吸出上清液。放置在盖和旋涡振荡板的底部。 </li><li>添加200μl的运行缓冲液，并重复在步骤10.14一次描述的洗涤步骤。 </li><li>添加95微升缓冲液中，然后用5微升7AAD每孔100微升的总体积/孔。孵育板在黑暗中在RT下15分钟。 </li><li>加入100微升缓冲液到每个孔中，并从板到集群管的每个孔中转移细胞悬浮液的整个体积，并获得对流动的样品仪。 </li><li>确定使用流式细胞分析软件7AAD阳性和CFSE阳性细胞的百分比。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+Regulatory+Science:+Research+to+Inform+Regulatory+Action+at+the+Food+and+Drug+Administration's+Center+for+Tobacco+Products.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cytotoxicity+of+different+tobacco+product+preparations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+cell-specific+cytotoxic+responses+of+oral+cavity+cells+to+tobacco+preparations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combustible+and+non-combustible+tobacco+product+preparations+differentially+regulate+human+peripheral+blood+mononuclear+cell+functions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+leukocyte+subsets+from+fresh+and+cryopreserved+peripheral+blood+mononuclear+cells+in+clinical+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dead+cell+discrimination+with+7-amino-actinomycin+D+in+combination+with+dual+color+immunofluorescence+in+single+laser+flow+cytometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multiparametric+flow+cytometry-based+cytotoxicity+assay+simultaneously+immunophenotypes+effector+cells:+comparisons+to+a+4+h+51Cr-release+assay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-induced+death+by+apoptosis+in+human+lymphokine-activated+natural+killer+cells.">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.

我们和其他人已经证实了外周血单个核细胞治疗用的TPP的抑制几种反应，包括表达和细胞因子和功能性的措施，如靶细胞杀死14的分泌。在以前的工作中描述的实验方法需要更长的潜伏期并且是温和的幅度14。鉴于这种诱人的体外模型的潜在应用基础研究和应用研究，我们调查是否有任何在这些多步生物分析测定的参数可以优化。在这里，我们提出了简化的方法来衡量使用TP...

知识点大幅身体的慢性吸烟的健康的不利影响，包括心血管疾病（CVD），慢性阻塞性肺疾病（COPD）和癌症1,2。慢性吸烟已经知道，引起炎症和免疫抑制，并且这些改变被报告给有助于微生物感染和癌症中的吸烟者3的危险性增加。 在体外和离体技术是有用在阐明的病理生理作用的分子基础香烟烟雾4-9（ 表1）和被认为是用于引导各种烟草制品10,11新兴调控的重要工具。 例如，我们已经表明，可燃烟草产品制剂（的TPP）如全烟雾条件培养基（WS-CM）和总颗粒物质（TPM）远远更多的细胞毒性和损害DNA比非易燃的TPP或尼古丁12,13。一贯以发表的作品，这是最近报道，可燃的TPP或尼古丁12,13。一贯以发表的作品，我们最近报道说，可燃的TPP造成显着的免疫抑制。这是证明了抑制长话样受体（TLR）-ligands，刺激细胞因子的分泌，并有针对性的细胞（K562）的体外模型14杀了外周血单个核细胞。鉴于炎症在香烟烟雾诱导的疾病过程的重要性，对测定条件进一步优化以评价香烟烟雾的免疫调节作用，提出本报告中。 体外测定法通常测量胞内和分泌的细胞因子，以及细胞毒性T细胞和NK细胞对K562细胞的细胞溶解作用杀伤测定法14。该试验涉及预孵化与WS-CM和尼古丁和PBMC的后续刺激s的TLR激动剂历时3天;最终读数是使用酶联免疫测定（ELISA）和/或流式细胞术进行。我们利用细菌脂多糖（LPS），其结合到TLR-4受体，并刺激的PBMC导致产生细胞内细胞因子和细胞因子的分泌。除了各测定步骤的优化，用于评估的TPP，我们还本方法分离的PBMC，细胞死亡测定法，和IL-8的量化的免疫调节作用。这些方法可应用于解决其他研究问题，并进一步细化，以评估烟草制品中的调节上下文。 表1.出版的体外报告和离体方法用于研究烟草产品制剂varilus病理生理作用的CS，香烟烟雾介质。 CSC，香烟烟雾冷凝水; CSE，香烟烟雾提取物; ELISA，酶联免疫吸附测定; GADPH，甘油醛-3-磷酸脱氢酶;定量PCR，定量聚合酶链反应; RT，实时定量聚合酶链式反应; TS，烟草烟雾。 <table><tbody><tr><td> 作者（研究年） </td><td> Laan 等人（2004） </td><td> 穆迪等人（2004） </td><td> 奥尔特曼斯等人（2005） </td><td> Vayssier（1998年） </td><td> Witherden 等人（2004） </td><td> 比勒尔等人（2008） </td></tr><tr><td> 使用电池 </td><td>人支气管内皮细胞（BEAS-2B），人中性粒细胞</td><td>人肺泡上皮细胞（A549） </td><td>人体气道平滑肌细胞（HASMC） </td><td>人类premonocytic U937细胞，人单核细胞</td><td> II型肺泡上皮细胞（ATII） </td><td>人单核细胞系（THP-1），人肺巨噬细胞</td></tr><tr><td> TPP使用 </td><td> CSE </td><td> CSC </td><td> CSE </td><td> TS </td><td> CSE </td><td> CS </td></tr><tr><td> 使用方法 </td><td> ELISA，定量PCR，迁移，电动汽车转变</td><td>免疫组织化学，电泳，Arrayscan试剂盒，RT-PCR法，酶联免疫吸附</td><td> ELISA法，RT-PCR，定量PCR，电泳</td><td>凝胶迁移率</td><td>光显微镜，电子显微镜，电泳，ELISA </td><td>定量PCR，ELISA，E-toxate试剂盒（Sigma），P65平板试验（TRANSAM），电泳，各种免疫试剂盒</td></tr><tr><td> 措施 </td><td> IL-8，GM-CF，AP-1，NF-κB，迁移</td><td>组蛋白乙酰转移酶，组蛋白脱乙酰酶，NF-κB，IL-8，等电点κB-α，GADPH </td><td> HO-1，GADPH，RANTES，IL-8，趋化因子</td><td>热休克/应激蛋白（HSP / HSP70），HF转录因子NF-κB，TNF-α的</td><td>表面活性蛋白（SP-A，SP-C），IL-8，MCP-1，GRO-α，TNF-α，IL-1β，IFN-γ的</td><td> IL-8，IL-1β，IL-6，TNF-α，MIP1-α，GRO-α，MAPK / JNK / ERK磷酸化，cJUN:DNA结合，谷胱甘肽，P65:DNA结合</td></tr><tr><td> 最终的结果 </td><td> CSE通过抑制AP-1的活化下调细胞因子的产生。 </td><td> 的 H 2 O 2与CSC提高组蛋白的乙酰化，减少组蛋白脱乙酰酶活性，差异调节促炎细胞因子的释放。 </td><td>香烟烟雾可能引起的IL-8从HASMC释放，增强由TNF-α，20％CSE更少的IL-8释放，趋化因子的抑制作用和RANTES由香烟烟雾。 </td><td> TS激活的HF的转录因子，其用的Hsp70过表达和抑制的NFkB的结合活性和TNF-α的释放相关联。 </td><td>减压ATII细胞衍生趋化因子水平的折衷肺泡拉尔修理，有助于香烟烟雾诱导的肺泡损伤和肺气肿。 </td><td>数据提供机械解释为什么吸烟者增加呼吸道感染。抑制先天应答的伴随增加的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

除其他病理生理改变，长期暴露于香烟烟雾导致炎症和免疫抑制，它已与吸烟者微生物感染和肿瘤发生率的增加的易感性。 体外抑制人外周血单核细胞中的受体介导的免疫应答（PBMCs）中与烟气成分处理是研究的机制和评估暴露于烟草制品的可能的长期影响的有吸引力的方法。在这里，我们的优化方法来执行体外使用的PBMC通过细菌脂多糖，Toll样受体4配体刺激的测定。整体烟条件培养基（WS-CM）的影响，可燃烟草制品的制备（TPP），和尼古丁通过在体外测定法的PBMC研究对细胞因子分泌和靶细胞杀伤。我们表明，分泌的细胞因子IFN-γ，TNF，IL-10，IL-6和IL-8和细胞内细胞因子IFN-47 ;, TNF-α和MIP-1α被压制在WS-CM-暴露的PBMC。效应器的PBMC的细胞毒性作用，如通过对K562靶细胞的杀伤测定法测定也减少通过暴露于WS-CM;尼古丁是效果最差的，这些检测。总之，我们提出了一组改进的测定法来评估的TPP的在体外测定法的影响，并且这些方法可容易地适用于测试其他感兴趣的产品。

结果以均数±标准平均（4供体样品）的错误。使用Excel软件以及t-检验对比进行与其对应的控制各处理，进行处理和未处理的对照样品之间的学生氏t检验。的统计学显着性指示的:*，P &lt;0.05; **，P &lt;0.005; *** P &lt;0.0005。 来测量暴露于WS-CM和尼古丁的影响，外周血单个核细胞分别用不同浓度的WS-CM和尼古丁3小时或24小时。细胞死亡是由坚固和可靠的7AAD染色法测定，7AAD插层成双链...

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.

方法评估细胞毒性的可燃烟草产品的准备和免疫抑制

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.4K 次观看.  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. 

视频: 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.

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

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

科研

医学

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.

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

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 ...

生物工程

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.

第三代机械 Vaping 装置生成电子烟气溶胶: 毒理学研究的应用

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

化学

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.

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

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.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

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

免疫与感染

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.

比较电子烟和烟雾在小说中的影响<em&gt;体内</em&gt;曝光系统

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

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

获取细胞毒性和细胞对生物材料的反应

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...

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.

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（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 ...

生物学

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

Source: Arimilli, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.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.

通过细胞内染色和流式细胞术分析烟草制品制剂对细胞因子产生的影响

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://appjove.unicartagenaproxy.elogim.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.

一种测量人精确切割肺切片中化学诱导的细胞毒性的测定法

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