This work was supported by EU FP7-ITN Marie-Curie Network program RADDEL. MB and KAJ would like to acknowledge Joe Varguese and Rui Serra Maia for their help and suggestions along the optimization of the Kupffer cell isolation.

所有的动物实验均符合所有相关的准则，法规和监管机构执行。所演示的协议英国内政部监管的指导和批准下进行 1.灌注和细胞采集（图1） <img alt="图1" src="/files/ftp_upload/52989/52989fig1.jpg" /> 图1:肝灌注的小鼠的麻醉之后，消化道横向移动到腹部的左侧，以使门静脉（PV）的访问。光伏使用的EGTA / HBSS溶液缓慢的流速（1-3毫升/分钟）和下veina静脉（IVC）是空心立即破裂，以避免肝脏内的任何超压。内灌注的第一分钟，流速逐渐增加至7毫升/分。胶原酶溶液，然后灌注以10毫升/分钟，直到其完全消化的实现。 <ol><li>编写，J再新鲜的材料表中描述的所有试剂。 </li><li>暖EGTA（乙二醇四乙酸）/ HBSS（Hank氏平衡盐溶液）溶液（每只小鼠50ml）和胶原酶溶液（每只小鼠100毫升）中30分钟，在40℃。 </li><li>首先用70％乙醇冲洗泵软管。倾40毫升EGTA / HBSS溶液到离心管浸在水浴中，并冲洗泵软管与预热EGTA / HBSS解。 </li><li>使用巴比妥类药物的呼吸抑制而死亡之前，可靠地生产无意识进行终端麻醉。苯巴比妥注射1毫克/千克，IP为女性或男性CD1小鼠（35-45克）。确认由麻醉捏脚趾。 </li><li>剃腹部毛和消毒用70％的乙醇溶液中的腹部表面。 </li><li>切断通过腹腔和通过横向移动肠道到腹部的左侧露出门静脉和下腔静脉。 </li><li>明星T中的泵以1-3毫升/分钟，EGTA / HBSS解决方案的速度和导管插入使用23克蝴蝶针门静脉（翅切）。夹住门静脉插管与23政针的部分，然后翻转serrefine镊子在打开小鼠腹部的表面上。肝脏应该在前30秒EGTA / HBSS解决方案灌注内迅速苍白。 </li><li>快速切割下腔静脉的下部，以避免在肝脏过量压力建筑物，再逐渐增加的流速至7毫升/分钟，在灌注的第一分钟。动物死亡是由于出血继发腔静脉穿刺。 </li><li>当降到低于5毫升EGTA / HBSS解决方案是将剩余的离心管，填补它与胶原酶溶液（40-50毫升）。逐渐增加的流速10毫升/分钟，在30秒内。 </li><li>通过将压力施加到下腔静脉 ，使用镊子，5-10秒的间隔使肝脏膨胀。 THIS能周期性地进行（在消化过程中的5-10倍）。此步骤会改善肝细胞​​解离，降低了灌注时间用胶原酶。 </li><li>当小于10毫升胶原酶溶液保留在离心管内，倒入40-50预热的胶原酶溶液放入离心管中。 </li><li>后灌注10-15分钟，约70-80毫升胶原酶溶液灌注，应用用钳子肝脏的表面上的小的压力。压力的打印指示肝细胞解离。 </li><li>从腹腔取出肝作为一件，并将其放置在含有20-30毫升库普弗细胞分离培养基的离心管中。保持肝细胞在冰上或4℃下进行3小时的最大时间段，以避免影响肝细胞活力。 </li><li>上重复，如果需要几个动物的灌注过程中，同时保持灌注肝脏在冰上。游泳池差一分三时肝脏净化并继续第3阶段。 </li></ol> 2.制备的密度梯度离心的（图2） <img alt="图2" src="/files/ftp_upload/52989/52989fig2.jpg" /> 图2:密度梯度的制备所有步骤均在无菌条件下进行。所述SIP通过混合15.3毫升涂布二氧化硅粒子的溶液1.7毫升10×PBS中制备。将5ml的SIP都用15毫升的PBS混合以赚20毫升25％的SIP溶液。 10毫升SIP都用10毫升的PBS混合以赚20毫升50％的SIP溶液。缓慢加入用血清学25毫升吸管含有50％的SIP溶液（20毫升）的倾斜，并使25％的SIP溶液（20毫升）的离心管中。 <ol><li>继续在无菌条件下以下步骤（部分2，3，5，和），并保持细胞在冰上或4℃。通过混合15.3毫升涂布二氧化硅粒子溶液1.7毫升10×PBS准备SIP（等渗包覆二氧化硅粒子溶液）。 </l我&gt; <li>为了赚20毫升25％的SIP解决方案，混合5毫升SIP用15毫升的PBS。为了赚20毫升50％的SIP解决方案，混合10毫升SIP用10毫升的PBS。 </li><li>填空的话就是离心管20毫升的50％的SIP解决方案。倾斜离心管以一个角度接近90°，在侧壁的管慢慢加入25％的SIP溶液（20毫升）用25毫升血清吸管，而不触及50％的SIP层的表面上。当加入25％的SIP溶液，逐步减少管的角度。保持密度梯度在冰上直到使用。 </li></ol> 3.库普弗细胞的纯化（图3） <img alt="图3" src="/files/ftp_upload/52989/52989fig3.jpg" /> 图3:库普弗细胞通过密度梯度离心和细胞粘附的纯化所有步骤均在无菌条件下进行。 （A）中的Glisson囊破裂后，肝细胞通过一个1过滤00微米的过滤器。将悬浮液离心×50克至丢弃肝细胞（粒料），并收集非实质细胞部分（上清液）。这个步骤重复3次。非实质细胞加入上不连续等渗梯度的顶部，并在800×g离心离心15分钟。从25％的SIP坐垫收集库普弗细胞通过细胞粘附的选择进一步纯化。 （B）的细胞接种于24孔板中并在37℃和5％CO 2的30分钟。非贴壁细胞用500微升的HBSS洗涤一次。 Kupffer细胞显示镀层，当f -CNTs可以加入到细胞中后，其贴壁形态学4小时。比例尺为25μm。 <a href="https://www.jove.com/files/ftp_upload/52989/52989fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>放一灌注肝脏在培养皿用15ml枯否细胞分离培养基。破裂格利森囊（用剪刀和释放所有肝细胞进入枯否细胞分离介质肝脏膜）。过滤溶液，虽然以被收集在一个离心管中100微米的细胞滤网。重复上附加灌注肝脏的灌注过程和（从三只小鼠从一个鼠标15毫升，高达45毫升）中汇集的细胞以相同的离心管中。 </li><li>离心细胞悬浮液，在50×g离心在4℃下2分钟。实质细胞（肝细胞）将在颗粒和非实质细胞（包括Kupffer细胞）将在上清液中。 </li><li>收集在一个干净的离心管中，离心上清，在50×g离心每2分钟。重复此步骤三次。 </li><li>离心机在1350×g离心15分钟以沉淀非实质细胞。弃上清，悬浮颗粒在10毫升枯否细胞分离培养基。 </li><li>添加的非实质细胞溶液的不连续等渗梯度五十分之二十五％，如在2.3中描述。一分钱rifuge在850 XG 15分钟没有加速或休息。 </li><li>本地化的25％的SIP分数接近25/50％SIP接口（ 图3）内出现混浊的富集库普弗细胞级分。使用10ml的血清吸液管，吸出约12毫升富集库普弗细胞级分的。转移细胞到含35-40毫升库普弗细胞分离培养基的离心管中。轻轻混匀离心机在1350×g离心15分钟，在4℃以沉淀细胞。 </li><li>丢弃在5-10毫升预热的枯否细胞培养基的上清液和重悬细胞。计数细胞（血球），并使用台盼蓝染色法测定存活率。 </li><li>板的纯化的非实质细胞在24孔板在5×10 5细胞/孔的密度。孵育细胞在37℃和5％的CO 2（ 图3）。 30分钟后，轻轻取出介质，洗涤预热Hank氏平衡盐溶液（HBSS）一次，然后更换它用500μl新鲜的和预温热库普弗细胞培养基（每孔）。 </li><li>离开Kupffer细胞至少4小时，在37℃和5％的CO 2治疗之前，纳米颗粒，以使它们能够获得他们附着的形态。 </li></ol> 4.表征<ol><li>枯否细胞纯度<ol><li>准备用于保持库普弗细胞活力新鲜的PBS / BSA溶液。用500μl的PBS / BSA溶液洗一次细胞。取出PBS / BSA溶液分离枯否细胞在500微升新鲜的PBS / BSA溶液用温柔刮擦。当细胞置于24孔板中，缩短刮刀的末端用剪刀，使脱离更容易进行。传递细胞成流式细胞仪管。 </li><li>计数使用血球和离心机1×10 5个细胞在1350×g下Kupffer细胞。重悬Kupffer细胞于30μlF4 / 80抗体（纯）的。拌匀，并在室温下孵育30分钟。保持在冰上的未染色细胞（未孵育的抗体）。 </li><li>加入2毫升的PBS / BSA溶液中染色的细胞，离心在1350 g下5分钟，弃去所得的上清液。悬浮在200μlPBS / BSA的染色细胞。 </li><li>用于流式细胞术分析，栅万根据它们的大小（前向散射）和粒度（侧向散射）不染色Kupffer细胞。在FL-1通道分析门控细胞的荧光。 </li><li>用同样的方法染色枯否氏细胞保持用于未染色术枯否细胞分析流程相同的设置。选择和量化染色细胞的荧光来评估枯否细胞的纯度。 </li></ol></li><li>枯否细胞吞噬活性<ol><li>用0.5％更换培养基（体积/体积）中的枯否细胞培养基的荧光珠孵育4小时，在37℃和5％的CO 2。 </li><li>为了在1350×g离心，二除去非内化珠，离心机Kupffer细胞SCARD上清，悬浮细胞在500μlPBS / BSA溶液。重复此步骤2次以上。 </li><li>刮枯否细胞在500微升PBS / BSA解决方案和转移的细胞成流式细胞仪管。 </li><li>离心机枯否细胞在1350 XG，丢弃在200微升PBS / BSA溶液的上清，重悬细胞。 </li><li>用于流式细胞术分析，栅万根据它们的大小（前向散射）和粒度（侧向散射）不染色Kupffer细胞。在FL-2通道门控分析细胞的荧光。 </li></ol></li></ol> 5.孵化化学官能化的碳纳米管（六-CNTs） <ol><li>通过超声处理在使用前15分钟，制备˚F-CNTs（1毫克/毫升）的水分散体:F -CNTs制备在内部22。 </li><li>准备2倍浓缩碳纳米管F- 分散枯否细胞培养基。 分散在介质中超声处理F-碳纳米管在进行步骤5.3前2分钟）。 </li><li>对于每个孔，用250微升2倍˚F-CNTs分散取代250微升旧媒体。未处理孔（250微升条件培养基+ 250微升新鲜库普弗细胞培养基的）中，用10％DMSO处理的孔用作阴性和阳性对照，分别为。 </li><li>孵育Kupffer细胞在37℃和5％CO 2的24或72小时。 </li></ol>在枯否细胞˚F-CNTs通过修改乳酸脱氢酶（LDH）测定6毒性评估<ol><li>弃去上清液。 </li><li>加入裂解缓冲液的200微升（每24孔板孔）并在37℃下进行30分钟-1小时。 </li><li>进行剧烈吸移后，收集裂解Kupffer细胞成离心管和离心机以40,000×g离心10分钟沉淀物溶解由细胞和细胞碎片使得f -CNTs。 </li><li>收集上清（ƒ-CNT免费）到离心管并在-20℃储存或继续执行步骤6.5）。 </li><li>转移50微升到96孔板中并加入底物混合物溶液（LDH试剂盒）等体积的各孔中。覆盖该平板，加入50μl的终止溶液（LDH试剂盒）之前在室温下孵育15分钟。添加含有50微升裂解缓冲液，50微升底物混合物溶液和50微升的终止溶液空白孔的一式三份。 </li><li>读取490nm处的吸光度在酶标仪并使用以下公式来计算（％）细胞活力。 </li></ol><img alt="式（1）" src="/files/ftp_upload/52989/52989eq1.JPG" />

<ol>
	<li>Hoffmann, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+novel+kidney+biomarkers+in+preclinical+toxicity+studies.">Performance of novel kidney biomarkers in preclinical toxicity studies.</a> Toxicol Sci. 116 (1), 8-22 (2010).</li><li>Nel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterial+Toxicity+Testing+in+the+21st+Century:+Use+of+a+Predictive+Toxicological+Approach+and+High-Throughput+Screening.">Nanomaterial Toxicity Testing in the 21st Century: Use of a Predictive Toxicological Approach and High-Throughput Screening.</a> Accounts of Chemical Research. 46 (3), 607-621 (2013).</li><li>Bregoli, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+antimony+trioxide+nanoparticles+on+human+hematopoietic+progenitor+cells+and+comparison+to+cell+lines.">Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines.</a> Toxicology. 262 (2), 121-129 (2009).</li><li>Yamasaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Aberrations+and+Cellular+Heterogeneity+in+SV40-Immortalized+Human+Corneal+Epithelial+Cells.">Genomic Aberrations and Cellular Heterogeneity in SV40-Immortalized Human Corneal Epithelial Cells.</a> Investigative Ophthalmology, &amp; Visual Science. 50 (2), 604-613 (2009).</li><li>Motoyoshi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megalin+contributes+to+the+early+injury+of+proximal+tubule+cells+during+nonselective+proteinuria.">Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria.</a> Kidney International. 74 (10), 1262-1269 (1038).</li><li>Prozialeck, W. C., Edwards, J. R., Lamar, P. C., Smith, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+barrier+characteristics+and+expression+of+cell+adhesion+molecules+in+proximal+tubule-derived+cell+lines+commonly+used+for+in+vitro+toxicity+studies.">Epithelial barrier characteristics and expression of cell adhesion molecules in proximal tubule-derived cell lines commonly used for in vitro toxicity studies.</a> Toxicology in Vitro. 20 (6), 942-953 (2006).</li><li>Chamberlain, L. M., Godek, M. L., Gonzalez-Juarrero, M., Grainger, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+non-equivalence+of+murine+(monocyte-)+macrophage+cells+in+biomaterial+and+inflammatory+models.">Phenotypic non-equivalence of murine (monocyte-) macrophage cells in biomaterial and inflammatory models.</a> J Biomed Mater Res A. 88 (4), 858-871 (2009).</li><li>Kermanizadeh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Kupffer+cells+in+the+hepatic+response+to+silver+nanoparticles.">The role of Kupffer cells in the hepatic response to silver nanoparticles.</a> Nanotoxicology. , (2013).</li><li>Fischer, H. C., Hauck, T. S., Gomez-Aristizabal, A., Chan, W. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+Primary+Liver+Macrophages+for+Studying+Quantum+Dot+Interactions+with+Biological+Systems.">Exploring Primary Liver Macrophages for Studying Quantum Dot Interactions with Biological Systems.</a> Advanced Materials. 22 (23), 2520-2524 (2010).</li><li>Wan, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M2+Kupffer+Cells+Promote+M1+Kupffer+Cell+Apoptosis:+A+Protective+Mechanism+Against+Alcoholic+and+Nonalcoholic+Fatty+Liver+Disease.">M2 Kupffer Cells Promote M1 Kupffer Cell Apoptosis: A Protective Mechanism Against Alcoholic and Nonalcoholic Fatty Liver Disease.</a> Hepatology. 59 (1), 130-142 (2014).</li><li>Matsuoka, M., Tsukamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+hepatic+lipocyte+collagen+production+by+Kupffer+cell-derived+transforming+growth+factor+&#946;:+Implication+for+a+pathogenetic+role+in+alcoholic+liver+fibrogenesis.">Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor &#946;: Implication for a pathogenetic role in alcoholic liver fibrogenesis.</a> Hepatology. 11 (4), 599-605 (1990).</li><li>Polakos, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kupffer+Cell-Dependent+Hepatitis+Occurs+during+Influenza+Infection.">Kupffer Cell-Dependent Hepatitis Occurs during Influenza Infection.</a> The American Journal of Pathology. 168 (4), 1169-1178 (2006).</li><li>Tanner, A., Keyhani, A., Reiner, R., Holdstock, G., Wright, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+enzymes+released+by+liver+macrophages+may+promote+hepatic+injury+in+a+rat+model+of+hepatic+damage.">Proteolytic enzymes released by liver macrophages may promote hepatic injury in a rat model of hepatic damage.</a> Gastroenterology. 80 (4), 647-654 (1981).</li><li>Knittel, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+matrix+metalloproteinases+and+their+inhibitors+in+parenchymal+and+non-parenchymal+cells+of+rat+liver:+regulation+by+TNF-&#945;+and+TGF-&#946;1.">Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-&#945; and TGF-&#946;1.</a> Journal of Hepatology. 30 (1), 48-60 (1999).</li><li>Branster, M. V., Morton, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Intact+Liver+Cells.">Isolation of Intact Liver Cells.</a> Nature. 180 (4597), 1283-1284 (1038).</li><li>Smedsr&#248;d, B., Pertoft, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+pure+hepatocytes+and+reticuloendothelial+cells+in+high+yield+from+a+single+rat+liver+by+means+of+Percoll+centrifugation+and+selective+adherence.">Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence.</a> Journal of Leukocyte Biology. 38 (2), 213-230 (1985).</li><li>Morin, O., Patry, P., Lafleur, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+endothelial+cells+of+adult+rat+liver+as+resolved+by+sedimentation+velocity+and+flow+cytometry.">Heterogeneity of endothelial cells of adult rat liver as resolved by sedimentation velocity and flow cytometry.</a> J Cell Physiol. 119 (3), 327-334 (1984).</li><li>Miller, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+cells+from+livers+of+carcinogen-treated+rats+by+free-flow+electrophoresis.">Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis.</a> Cancer Res. 43 (9), 4176-4179 (1983).</li><li>Firme, C. P., Bandaru, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+issues+in+the+application+of+carbon+nanotubes+to+biological+systems.">Toxicity issues in the application of carbon nanotubes to biological systems.</a> Nanomedicine. 6 (2), 245-256 (2010).</li><li>Ali-Boucetta, H., Al-Jamal, K. T., Kostarelos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+assessment+of+carbon+nanotube+interaction+with+cell+cultures.">Cytotoxic assessment of carbon nanotube interaction with cell cultures.</a> Methods in Molecular Biology. 726, 299-312 (2011).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+and+correcting+for+carbon+nanotube+interferences+with+a+commercial+LDH+cytotoxicity+assay.">Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay.</a> Toxicology. 299 (2-3), 99-111 (2012).</li><li>Wang, J. T. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetically+Decorated+Multiwalled+Carbon+Nanotubes+as+Dual+MRI+and+SPECT+Contrast+Agents.">Magnetically Decorated Multiwalled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents.</a> Advanced Functional Materials. 24 (13), 1880-1894 (2014).</li><li>Li, P. Z., Li, J. Z., Li, M., Gong, J. P., He, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+to+isolate+and+culture+mouse+Kupffer+cells.">An efficient method to isolate and culture mouse Kupffer cells.</a> Immunol Lett. 158 (1-2), 52-56 (2014).</li><li>Froh, M., Konno, A., Thurman, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+liver+Kupffer+cells.">Isolation of liver Kupffer cells.</a> Curr Protoc Toxicol. Chapter 14, Unit14 14 (2003).</li></ol>

The authors declare that they have no competing financial interests.

下面的步骤是关键的，以实现高产量和枯否氏细胞的高存活率。在无菌条件应用于限制细菌和真菌污染的风险。所有仪器使用前需要灭菌。试剂应在进行分离步骤之前新鲜制备。 Ⅳ型胶原酶的选择，具有低胰蛋白酶的活性，是至关重要的。不同批次来自同一供应商具有不同的酶活性和它们可能需要最初比较，以便选择最合适的批次库普弗细胞分离。这可以通过评估细胞产量?...

纳米毒理学研究领域的目的是表征纳米颗粒的生物效应。基于在体内研究毒理学研究仍然是最精确的方法。然而，它们的使用受到它们的成本，劳动和时间要求1限定。作为替代， 在体外测定法已被使用，因为它们的简单性和开发高通量体外测试平台2的可能性-如此延伸的测试条件的数量。最nanotoxicological研究使用体外测定与永生化的细胞系进行。然而，也有关于这些实验的结果外推到体内毒理学影响3担忧。事实上，永生化细胞系的性质可以是显著不同从组织它们衍生自， 例如 ，遗传转化4，关键形态特征恶化5，细胞极性6和功能的改变，如炎症介质7的调节的丧失。 Kupffer细胞是最丰富的巨噬细胞群中的主体，并直接与血液接触由衬肝血窦的壁上。作为网状内皮系统（RES）的一部分，这些巨噬细胞是负责循环纳米颗粒和因此的捕获，构成一个非常合适的模型来研究纳米颗粒的毒性。 体内 8 并在体外与暴露于纳米粒子枯否氏细胞相关的炎症反应9研究已在其他地方发表。枯否细胞也参与肝脏疾病的发病机制，如酒精性肝病10，肝纤维化11或病毒性肝炎12。据报道，孤立的枯否细胞提供了有益的启示来形容细胞机制INVolved肝破裂13,14。 几种方法已经报道了分离和纯化Kupffer细胞。细胞的分离可导致机械或酶解15。胶原酶消化节约表明枯否细胞的功能完整性，而导致高枯否细胞产量16的优势。许多实验方法，在复杂性和成本不同，已用于Kupffer细胞从其他肝细胞群中分离出来。例如，枯否细胞纯度可通过免疫亲和17，流电泳18，选择性粘附16或通过离心技术16，根据它们的大小和密度而选择的细胞来实现。这些方法的组合可以被选择为增加的人口16的纯度。没有关于对枯否细胞分离的理想方法共识的，因为它主要取决于应用和可用的equipmen吨。然而，在纳米颗粒的毒性测试的情况下，简单性和高收率与Kupffer细胞的功能保存相关的技术似乎是对本申请中最适合的。 这里报道的枯否细胞的分离方法是基于一种两步灌注法进行纯化上密度梯度。该方法是从由Smedsrød 等人开发的原始协议进行修改。16设计用于大鼠肝细胞的分离。大多数研究报告，并从大鼠肝脏描述Kupffer细胞的分离。在此，我们描述了一种方法，从小鼠肝脏分离Kupffer细胞，以高收率和纯度。使用小鼠降低了实验成本，并允许几个肝脏的处理，以获得纳米颗粒的毒性测试大量Kupffer细胞。 在下面的协议，枯否细胞与官能化的碳纳米管（六</EM&gt; -CNTs）。碳纳米管的独特的物理化学性质- 即高长径比和表面积大，都取得了碳纳米管有趣候选作为用于治疗和诊断目的的载体。然而，忧虑已提出了关于碳纳米管19的毒性，以及新的体外测试的发展旨在提高CNT生物效应的理解。在枯否细胞毒性与缺乏细胞膜的结构完整性有关。这是通过胞质酶LDH的损失从细胞到上清液计量。这种方法的原理，因此，是除去任何释放LDH和测量什么留在电池20。这在要求完成测量所释放的LDH在上清液因为CNT的上清液中的存在干扰测定21。 我们建议使用这种简单和成本效益枯否细胞的分离实现方法具的d，来分离大量的功能枯否细胞。这允许范围的纳米颗粒的毒性的筛选，在一个相关的初级巨噬细胞的模型。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td dir="LTR">Euthatal (pentobarbital sodium)</td>
			<td dir="LTR">Merial</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
		</tr>
		<tr>
			<td dir="LTR">CD1 mice</td>
			<td dir="LTR">Charles River&#160;&#160;</td>
			<td dir="LTR">-</td>
			<td dir="LTR">Mouse weight should vary from 35 to 45 g. We advise the use of male CD1 mice as their weight increase rapidly (e.g. a male CD1 mouse of 7 weeks old exceeds 35 g in weight). It is also advised to contact the animal supplier in advance to arrange for the delivery of older animals. Alternatively animals can be in-house to reach the desired weight.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">HBSS (Ca2+ and Mg2+ free, with bicarbonate)&#160;&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">14175-053</td>
			<td dir="LTR">HBSS must be&#160; Ca2+ free.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Ethylene Glycol Tetraacetic Acid (EGTA) tetrasodium salt&#160;</td>
			<td dir="LTR">Sigma-Aldrich&#160;&#160;</td>
			<td dir="LTR">E8145&#160;&#160;</td>
			<td dir="LTR">EDTA can also be used but EGTA has the advantage of chelating Ca2+ selectively.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">HEPES (1M)&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">15630-056&#160;</td>
			<td dir="LTR">100 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Collagenase type IV&#160;</td>
			<td dir="LTR">Worthington&#160;</td>
			<td dir="LTR">CSL-4&#160;</td>
			<td dir="LTR">It is advised to test different batches of collagenase or at least mention to the supplier the product has to be suitable for liver cell isolation&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">&#160;Low glucose DMEM&#160;</td>
			<td dir="LTR">Sigma-Aldrich&#160;&#160;</td>
			<td dir="LTR">D5523&#160;</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">RPMI (with sodium pyruvate and Glutamax&#174;)&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">12633-012&#160;</td>
			<td dir="LTR">500 ml</td>
		</tr>
		<tr>
			<td dir="LTR">Penicillin/Steptomycin&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">15140-122</td>
			<td dir="LTR">100 ml</td>
		</tr>
		<tr>
			<td dir="LTR">Fetal Bovine Serum&#160;</td>
			<td dir="LTR">First-Link&#160;&#160;</td>
			<td dir="LTR">60-00-850&#160;</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Trypan blue solution&#160;</td>
			<td dir="LTR">Sigma-Aldrich&#160;</td>
			<td dir="LTR">T8154&#160;</td>
			<td dir="LTR">100 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Coated silica particle solution (Percoll&#174;)</td>
			<td dir="LTR">GE Healtcare&#160;&#160;</td>
			<td dir="LTR">17-0891-02&#160;</td>
			<td dir="LTR">Percoll&#174; is very stable and can be kept for several years&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">1x Phosphate Buffered Saline&#160;&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">10010-023</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">10x Phosphate Buffered Saline&#160;&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">70011-036</td>
			<td dir="LTR">500 ml</td>
		</tr>
		<tr>
			<td dir="LTR">Dimethyl sulfoxide&#160;</td>
			<td dir="LTR">Fisher&#160;</td>
			<td dir="LTR">D/4120/PB08&#160;</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">LDH kit (CytoTox 96&#174;)&#160;</td>
			<td dir="LTR">Promega&#160;</td>
			<td dir="LTR">G1781&#160;</td>
			<td dir="LTR">Keep protected from light&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">DMEM phenol red free</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">31053-028&#160;</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">F4/80 antibody (Alexa 488)</td>
			<td dir="LTR">AbD Serotec</td>
			<td dir="LTR">MCA497A488</td>
			<td dir="LTR">Do not dilute, used neat for flow cytometry</td>
		</tr>
		<tr>
			<td dir="LTR">Fluorescent beads</td>
			<td dir="LTR">Sigma</td>
			<td dir="LTR">L2778</td>
			<td dir="LTR">Latex beads, amine-modified polystyrene, fluorescent red. 1 ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name of the Material</td>
			<td>Company&#160;</td>
			<td>Catalog number&#160;</td>
			<td>Comments/Description&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Butterfly blood collection set (23G/305mm long tubing)&#160;&#160;</td>
			<td dir="LTR">BD&#160;</td>
			<td dir="LTR">367288</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Syringe Filters (0.22 &#956;m Blue Rim)&#160;</td>
			<td dir="LTR">Minisart&#160;</td>
			<td dir="LTR">16534-K&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Centrifuge Tubes (50 ml Blue Cap)&#160;</td>
			<td dir="LTR">BD Biosciences, Falcon&#160;</td>
			<td dir="LTR">35 2070&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Petri dish (90 x 15 mm)&#160;</td>
			<td dir="LTR">Thermo Fisher Scientific&#160;&#160;</td>
			<td dir="LTR">BSN 101VR20&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">100 &#956;m cell strainer&#160;&#160;</td>
			<td dir="LTR">BD&#160;&#160;</td>
			<td dir="LTR">352360</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Peristaltic pump&#160;</td>
			<td dir="LTR">Watson Marlow&#160;</td>
			<td dir="LTR">SciQ 300&#160;&#160;</td>
			<td>Rinse tubing before and after each usage with sterile PBS and 70% ethanol.</td>
		</tr>
		<tr>
			<td dir="LTR">24-well plates</td>
			<td dir="LTR">Corning&#160;&#160;</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">96-well plates&#160;</td>
			<td dir="LTR">Corning&#160;&#160;</td>
			<td dir="LTR">3595</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Plate reader</td>
			<td>BMG Labtech</td>
			<td>FLUOstar Omega</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Serrefine forceps</td>
			<td>Hammacher GmbH&#160;</td>
			<td>Art. Nr. HSE 004-35 / Cat. Nr. 221-0051</td>
			<td>The serrefine forceps allow to clamp the vessel cannulated with the 23G needle without the need of holding the forceps during the perfusion procedure. URL: (http://www.hammacher.de/Laboratory-Products/Clamps-forceps/Serrefines/HSE-004-35-Serrefine::25126.html)&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Flow cytometry tubes</td>
			<td>BD Biosciences, Falcon&#160;</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Microcentrifuge tubes (1.5 ml)</td>
			<td>Elkay</td>
			<td>&#160; 000-MICR-150</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Cell scraper</td>
			<td>BD Biosciences, Falcon</td>
			<td>353086</td>
			<td>Cut blade extremities with a pair of scissors to scrape cells in 24-well plates.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Name of the Reagent</td>
			<td dir="LTR">Company&#160;</td>
			<td dir="LTR">Catalog number&#160;</td>
			<td dir="LTR">Comments/Description&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">&#160;EGTA (Ethylene Glycol Tetraacetic Acid)/HBSS (Hank&#39;s Balanced Salt Solution) Solution&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">HBSS containing 0.5 mM EGTA and 25 mM HEPES. Adjust pH to 7.4. Prepare 50 ml for each liver to perfuse.&#160;&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Collagenase Solution&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">DMEM low glucose containing collagenase type IV at 100 UI/ml, 15 mM HEPES and 1% Penicllin/Streptamycin (v/v). Adjust pH to 7.4. Prepare 100 ml for each liver to perfuse. After adding the collagenase, it is advised to warm up the solution for 30 min before use. This allows the collagenase activity to be optimum.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Kupffer Cell Isolation Medium&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">RPMI containing, 1% Non-Essential Amino-Acids (v/v),1% glutamax&#174; (v/v) and 1% Penicllin/Streptomycin (v/v). Prepare at least 100 ml for 1-3 livers.&#160;&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Kupffer Cell Culture Medium&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">RPMI containing 10% Fetal Bovine Serum, 1% Non-Essential Amino-Acids (v/v),1% Glutamax&#174; (v/v) and 1% Penicllin/Streptamycin (v/v). Prepare at least 100 ml for 1-3 livers.&#160;&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">SIP (solution of isotonic coated silica particles)</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">Mix 1.7 ml of 10x Phosphate Buffered Saline&#160; with 15.3 ml of Percoll&#174; to obtain 17mL of SIP.</td>
		</tr>
		<tr>
			<td dir="LTR">25% SIP solution&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">Mix 5 ml of SIP with 15 ml of 1x Phosphate Buffered Saline&#160;&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">50% SIP solution&#160;</td>
			<td dir="LTR"></td>
			<td></td>
			<td dir="LTR">Mix 10 ml of SIP with 10 ml of 1x Phosphate Buffered Saline&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Lysis Buffer</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">DMEM media with 0.9% Triton X-100</td>
		</tr>
		<tr>
			<td dir="LTR">PBS/BSA Solution</td>
			<td dir="LTR"></td>
			<td></td>
			<td dir="LTR">Prepare fresh Phosphate Buffered Saline pH 7.4 with 1% Bovine Serum Albumin.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
		</tr>
	</tbody>
</table>

kupffer cell isolation for nanoparticle toxicity testing

绝大多数体外 nanotoxicological研究已经使用永生化细胞系为他们的实用性。然而，从纳米颗粒的毒性测试结果在永生化细胞系或原代细胞已经显示不符，强调需要延长使用的原代细胞的体外测定法。这个协议描述了小鼠肝巨噬细胞的分离，命名Kupffer细胞，以及它们的使用来研究纳米颗粒的毒性。 Kupffer细胞是最丰富的巨噬细胞群在体内和构成网状内皮系统（RES），负责循环纳米颗粒的捕获部分。这里报道的枯否细胞的分离方法是基于一种两步灌注法进行纯化上密度梯度。该方法的基础上，胶原酶消化和密度离心，适于从由Smedsrød 等人开发的原始协议。专为大鼠肝细胞的隔离和n和提供高产率（每鼠14×10 6个细胞）和枯否细胞的高纯度（&gt; 95％）。这种隔离方法不需要复杂的或昂贵的设备，因此代表的复杂性和细胞产量之间的理想折衷。使用较重的小鼠（35-45克）提高了分离方法的产量，但也有利于显着的门静脉插管的过程。的官能化的碳纳米管˚F-CNTs毒性测定在该模型由改性的LDH测定。该方法通过测量缺乏的枯否细胞膜结构完整性孵育以f -CNTs后评估细胞生存力。毒性反应为f -CNTs可以使用一致此法来衡量，强调指出，孤立的枯否细胞对纳米颗粒的毒性测试非常有用。纳米毒理学的整体理解可以受益于这种模式，使得纳米颗粒的选择临床翻译更多艾菲cient。

非纯化的实质细胞的数目是一致的和远程每只小鼠之间8和14×10 6个细胞（17隔离被执行）。每只小鼠隔离足以板16〜28的孔中。通过台盼蓝染色细胞存活率显示出细胞存活率〜95％。库普弗细胞显示内温育30分钟的圆形状，在37℃，与它们的不完全粘合的形态（ 图4A）。在4小时温育，之后，将细胞分散和细胞簇开始形成。 然后枯否细胞的特点，以确认其纯度...

Watch this Scientific Journal Video about Kupffer Cell Isolation for Nanoparticle Toxicity Testing at JoVE.com

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

枯否细胞分离的纳米粒子毒性测试

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

kupffer-cell-isolation-for-nanoparticle-toxicity-testing

Research

JoVE Journal

Immunology and Infection

23.6K 次观看.  King's College London. Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells. 

视频: Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

科研

免疫与感染

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...