作者这项工作是由NIH / NCI补助＃R01CA172138（与SBE）的支持。

涉及受试动物的程序已批准的机构动物护理和使用委员会（IACUC）在特拉维夫大学。 1.鼠协议<ol><li>准备工作<ol><li>安排2蝶形21g的针头。或者，通过提交针的锋利边缘准备2钝刃蝴蝶21g的针头。 </li><li>消毒手术工具:2对剪刀，钝边镊子，止血钳，齿形组织钳通过高压釜在121℃下对重力（干燥）设定至少30分钟灭菌。 </li><li>通过加入每毫升磷酸盐缓冲的盐水（1×PBS）溶液肝素30单位制备肝素的PBS（30单位/ ml）。使用在室温。 35毫升是每只动物所需的最小体积。 </li><li>流肝素的PBS放入蠕动泵线和蝴蝶针以避免气泡。 </li></ol></li><li> MP-白细胞的肺和收集灌注<ol><li> 8％的异氟醚过量安乐死的动物。通过确认呼吸监测停止安乐死。 </li><li>当呼吸停止，立即打开使用无菌剪刀和牙齿组织钳腹腔和胸腔腔。执行沿腹部到剑突中线腹部切口。 </li><li>解除使用镊子，小心地切割所述左肋两侧胸骨，没有穿刺任何内部器官或大血管。夹住胸骨止血，并吻侧折肋骨露出心肺复杂。 </li><li>插入一个蝴蝶针插入心脏的右心室，大约在一分钟内采集的血液量最大到注射器（〜从250克动物6ml）中。 </li><li>夹紧腔静脉用止血钳来避免肝脏（见讨论 ）落后灌注。 </li><li>右心室内保持蝶形针而更换含血液与p的流出管的注射器eristaltic泵。 </li><li>插入连接于5ml的收割注射器进入左心室的第二蝶形针，避免了室间隔渗透。 </li><li>打开蠕动泵以大约5毫升/分钟的速度，并轻轻收集到注射器被污染的血液灌流的第一毫升。继续，直到灌洗液颜色由深变为淡红色。 （丢弃血液污染perfuste）。 </li><li>无蠕动泵停止，快速替换20ml的收割注射器中的5毫升收获注射器和收集20毫升采用灌注的更高的速度（最多7毫升/分）肺灌注液。连续监视灌注液流入收集注射器，同时避免真空的形成。 </li><li>停止蠕动泵的流动。 </li></ol></li><li>提取白细胞<ol><li>离心灌注液在400×g下10分钟。 </li><li>吸出流体。 </li><li>加入10毫升的PBS或培养基，离心，在400×g离心10分钟，吸出流体。重复此步骤两次。使用PBS或培养基的依赖于随后使用的样本。 </li><li>加入20毫升的PBS或培养基，离心，在400×g离心10分钟，吸出流体。 </li><li>重构该细胞至所需浓度，基于样品使用（FACS，PCR 等 ）。 </li></ol></li></ol> 2.鼠标协议<ol><li>准备工作<ol><li>安排2蝶形地下25针。或者，通过提交针的锋利边缘准备2钝刃蝴蝶地下25针。 </li><li>消毒手术工具:2对剪刀，钝边镊子，止血钳，齿形组织钳通过高压釜在121℃下对重力（干燥）设定至少30分钟灭菌。 </li><li>通过加入每毫升1×PBS溶液肝素30单位制备肝素的PBS（30单位/ ml）。使用在室温。 25毫升是每只动物所需的最小体积。 </li><li> STRE正在肝素的PBS放入蠕动泵线和蝴蝶针以避免气泡。 </li></ol></li><li> MP-白细胞的肺和收集灌注<ol><li> 8％的异氟醚过量安乐死的动物。通过确认呼吸监测停止安乐死。 </li><li>当呼吸停止，立即打开使用无菌剪刀和牙齿组织钳腹腔和胸腔腔。执行沿腹部到剑突中线腹部切口。 </li><li>解除使用镊子，小心地切割所述左肋两侧胸骨，没有穿刺任何内部器官或大血管。夹住胸骨止血，并吻侧折肋骨露出心肺复杂。 </li><li>夹紧腔静脉用止血钳来避免肝脏（见讨论）落后灌注。 </li><li>插入连接到所述蠕动泵的流出管蝶形针进入右VEN心脏的tricle，并连接到新的2 ml注射器采集到心脏，室间隔avoidingpenetration的左心室第二蝴蝶针。 </li><li>打开泵的约2毫升/分钟的速度，并收集血液污染的灌流液的第一毫升到收割注射器直至灌注液从黑变为淡红色（丢弃血液污染perfuste）。 </li><li>无蠕动泵停止，快速替换10ml的收割注射器2 mL萃取收获注射器和收集10毫升采用灌注的更高的速度（最多4毫升/分钟）肺灌注液。连续监视灌注液流入收集注射器，同时避免真空的形成。 </li><li>停止蠕动泵的流动。 </li></ol></li><li>提取白细胞<ol><li>离心灌注液在400×g下10分钟。 </li><li>吸出流体。 </li><li>加入10毫升的PBS或培养基，离心，在400xg离心10分钟，吸出流体。重复此步骤三次。使用PBS或培养基的依赖于随后使用的样本。 </li><li>加入10毫升的PBS或培养基，离心，在400×g离心10分钟，吸出流体。 </li><li>重构该细胞至所需浓度，基于样品使用（FACS，PCR 等 ）。 </li></ol></li></ol>

<ol>
	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, M., 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+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunol Res. 58, 28-39 (2014).</li><li>Melamed, R., 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+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Shakhar, 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=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><li>Melamed, R., 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=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, 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=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

作者什么都没有透露。

本文提供的方法使得能够选择性收获和MP-白细胞的独特群体的研究。相比循环或脾的白细胞中，MP人口的特征在于白细胞亚群的一个独特的组合物，更高的活化水平，各种细胞因子的更高的释放，和亲和抗炎细胞因子2,3的更高的mRNA水平。具体地，我们已经表明，MP-NK细胞比循环NK细胞对各种靶细胞3,4-多种细胞毒性，并表达更高水平的干扰素γ的。总的来说，我们的结果表明，MP-NK白细胞具有比循环NK细胞，并因此可能意味着在控制血源性转移和肺转移更大的生物学意义更大的细胞毒性。因此，脾或循环白细胞不应当被认为是金标准或用于反射免疫状态默认人口，和人类的研究，玉米monly仅仅依靠循环中的白细胞，应谨慎解释。此外，由于各种疾病特别是造成肺，免疫应本机关内的研究，和MP室应该从质，实质和野马肺泡副舱室进行区分，事实上是用我们的方法灌注的情况。 为了保证此过程的成功，这是与（i）打开腹部和胸部腔迅速以下呼吸完全停止，以维持血压，使最大心采血，和（ii）从一个不希望的额外避免关键通过使用钝边的蝴蝶针的心脏心室穿孔。另一个关键障碍是血的肺灌洗液中的潜在污染。肺灌洗液的第一毫升被污染心脏血，这表现它们应被丢弃。为了实现ANI之间的标准化MALS，开始收集在MP灌洗液的标准是基于从暗红色转移到淡红色。此外，建议使用非锯齿止血夹紧腔静脉 ，避免了肝脏的向后灌注，特别是如果希望还收集通过肝脏的连续灌注marginating肝细胞。同时收集从左心室灌注液，关键的是不穿透室间隔。最后，用于灌注在PBS中肝素是用于从毛细管断开MP-白细胞种群至关重要的，因此必须使用。 执行此过程时，可能会出现一些故障，但其中大部分都是可纠正的。如果从针头插入心脏的部位渗透泄漏，建议使用钝头镊子与周围的蝴蝶针锯齿技巧封住破裂心肌。如果针头滑出，申通快递P中的蠕动泵，重新插入针头插入原来的渗透网站，并重新启动水泵。如果收集流动停止，停止收集真空压力，心室其他地方针，然后重新启动集合。如果左心房膨胀，并且不被连续的集合减少，降低了泵的流速。在一般情况下，高的蠕动流量可能会损害毛细管形成，以及肺中，可能变得肿胀。 本文所描述的方法使得在MP白细胞种群的选择性收获以相对简单的过程。考虑到这部分人群表现出相较于其他免疫车厢白细胞独特的特点，而这一人群可能有生物体的生物及临床意义，我们特别研究肺相关的进程和疾病时可行时，建议使用此方法。它是目前未知是否MP细胞，其驻留在肺capill白羊在细胞收获，已经成熟在肺，或他们是否会保持在离开肺及其特性。如从其他车厢白细胞可能不反映在MP人口的轮廓，并且由于不是在人类可访问的，我们还建议使用这种方法来阐明特定循环生物标志物，与独特的MP特征相关。

白细胞粘附到肺部的毛细血管（ 即 marginating肺（MP），白细胞）1被示出与来自其他免疫区室（ 例如 ，循环，脾，骨髓）的白细胞显示出不同的白细胞组成和独特活性2- 4。例如，MP-白细胞表现出较高的天然杀伤（NK）细胞的细胞毒性对各种肿瘤细胞相比，循环和脾NK细胞，以及分化信使RNA（mRNA）水平和升高亲和抗炎性细胞因子的分泌。细胞的组合物也由循环白细胞分化为MP-白细胞具有相比循环白细胞（50％对30％，）先天/适应性免疫的比例较高。本文提出的方法的目标是使MP-白细胞选择性收割，为了研究这个重要和独特的免疫隔室（细胞群），以及elucIDATE对这些特定单元的各种操作（ 例如 ，免疫激活）的影响。 为了理解这个独特群体的意义，要注意的是，免疫系统能够控制通过细胞介导的免疫（CMI）的体内功能循环肿瘤细胞，微转移和残余疾病是很重要的。尽管免疫系统的先例未能控制原发肿瘤这种能力是显而易见的，并且在癌症患者和动物模型5 的体内证据充分的支持。重要的是，这些研究结果通常与在体外和离体研究，其中报告，大多数自体肿瘤细胞是抗细胞毒性通过从人类和动物的循环血液样品中的白细胞不一致（由细胞毒性测定法测定）6,7。这种差异可能是由于不同的白细胞群，如前述的体内存在上述的MP白细胞种群，特别是其活化NK细胞3的亚群。实际上，同基因的肿瘤细胞（MADB106），这被认为是对循环和脾白细胞抗性，却显示出了由MP-NK细胞-3,8-被裂解。因此，转移至的fischer344（F344）大鼠肺中据称"NK抗性&#39;MADB106细胞通过MP-NK细胞，但不被循环或脾NK细胞，其通常研究了给予它们易于访问的控制。 纯化和活性的MP-白细胞通过从肺，其基于肺组织研磨或生物降解9白细胞的标准收获方式无法访问。相比于组织处理方法我们的方法有两个主要的优点。首先，将灌注方法选择性地收割的MP-白细胞，来自其他小区从肺实质，间质性起源，和支气管肺泡COMPAR将它们分离tments。其次，灌注技术更好保持完整性和MP-白细胞的生理环境，不像研磨和生物处理方法损害细胞，改变它们的形态，并诱导生产和调节免疫活性和特异性地抑制NK细胞的各种因素释放细胞毒性10。 肺是癌症转移和用于各种感染性疾病的主要靶器官。所有的循环恶性细胞和感染细胞穿过肺毛细血管，在那里他们需要变形和毛细血管内皮细胞和白细胞居民互动。在这些条件下，循环细胞可以容易地由驻留MP-白细胞定位。因此它似乎有活化的白细胞在这种免疫隔室生物学有利的，并且在不同的生物，实验和临床研究这种独特的MP-人口是重要的。这是值得要注意，SY通过各种生物响应改性剂stemic免疫激活（ 例如 ，聚肌胞苷酸（聚I:C）或C型的CpG寡脱氧核苷酸（CpG基-C ODN））已经显示激活MP-白细胞比循环白细胞3以上， 8,11。

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Heparin sodium porcine mucosa (perservative free)</td><td>Sigma Aldrich</td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-pulmonary leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

在MP-车厢表现出不同的白细胞子集组成相比，循环中的白细胞。使用流式细胞仪分析，白细胞亚群被确定和量化来表征两个循环和MP白细胞的组合物。粒细胞和淋巴细胞基于前向和侧向散射进行了鉴定。淋巴细胞内，NKRP-1 亮细胞被鉴定为NK细胞，CD3 + T细胞，RM1 +作为单核细胞，和CD4 + / CD8 + / CD25 +被用于T细胞亚群的识别。先天免疫细胞，包括粒细胞，单核细胞，和NK细胞，构成的MP-白细胞人口的52％（±1.52％）相比，在循环中（P &lt;0.05）27.9％（±0.66％）。相反，T细胞亚群，包括CD4 +，CD4 + CD8 + T细胞和监管（CD4 + CD25 +）T细胞，以及树突状细胞相比，流通在多功能隔室显示出较低百分比（P &lt;0.005; N = 8）。在该菌株的MP的隔室的细胞产量构成的约300万的白细胞（ 图1）。 内NK细胞，两个亚群是显而易见的，基于小区大小。利用NKRP-1贴标前向散射的散点图，NK细胞分为大大小小的NK细胞。在MP隔室显示出相比于循环（约10％），大的NK细胞（〜30％）的3倍更高的百分比，而NK细胞的总数是相似的（ 图2）。此外，使用标准的4小时51 Cr释放测定，对若干靶肿瘤细胞的NK抗肿瘤的细胞毒性，测定8不久，该测定评价51的Cr从肿瘤细胞释放由NK细胞的特定的细胞毒性的结果的量秒。 NK细胞从MP隔室以裂解肿瘤细胞的能力比循环NK细胞（ 图3），针对两个MADB106同基因细胞系（ 图3A），为明显更大，并且YAC-1标准细胞系（ 图3B） 。此外， 通过体内多聚免疫刺激我:C管理产生比上流传的NK细胞时MADB106靶细胞系被使用（ 图3A）的MP-NK细胞的NK细胞活性更深远的影响。 <img alt="图1" src="/files/ftp_upload/53849/53849fig1.jpg" /> 相比于F344大鼠循环图1.不同白细胞子集组成表征MP-舱 。在多功能隔室，先天免疫的馏分较大相比，其当量在循环（52％对28％），而T细胞的患病率表现出相反的差异（p0; 0.005; N = 8）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53849/53849fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53849/53849fig2.jpg" /> 图2.增加大NK细胞的百分比在多功能隔室相比，循环的MP-NK细胞表现出三倍，分别比循环NK细胞大NK细胞的比例更高（30％比10％; P &lt; 0.05），NK细胞的总数量是相似的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53849/53849fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53849/53849fig3.jpg" /> 图3.在多功能隔室高等NK细胞的细胞毒性相比，流通。（A）MADB106靶细胞-从MP车厢收获NK细胞表现出对这种细胞有明显的细胞毒作用，而循环NK细胞不显示细胞毒性在体内多聚免疫刺激我:C增加了MP车厢NK细胞活性，但不在循环。 （B）YAC-1靶细胞-相比，循环NK细胞对这种标准的细胞系从MP车厢收获NK细胞表现出较高的细胞毒性。由聚免疫刺激I:C增加在两个MP隔室和循环NK细胞的细胞毒性。 NK细胞数目控制为细胞毒性的评估。数据表示为平均值±SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53849/53849fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating肺白细胞的选择性收获

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

selective-harvesting-of-marginating-pulmonary-leukocytes

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.8K 次观看.  Tel-Aviv University. Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

视频: Selective Harvesting of Marginating-pulmonary Leukocytes

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

科研

免疫与感染

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating肝白细胞的选择性收获

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

右心导管插入术后天鹅管囊小贴中内皮细胞的提取

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

医学

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

<meta charset="utf8"></head><body>从小鼠肺中分离免疫和非免疫细胞，用于流式细胞分析

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

<meta charset="utf8"></head><body>从小鼠肺中分离活髓细胞和上皮细胞群

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

从小鼠肺中分离活髓细胞和上皮细胞群

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

使用磁性细胞选择建立体外 LLEC 培养物

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

作者聚焦：揭示软脑膜淋巴内皮细胞的细胞功能和潜在临床意义 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

软脑膜淋巴内皮细胞的收获和原代培养

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

Marginating肺白细胞的选择性收获

摘要

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此视频中的章节

小鼠肺树突状细胞的分离

Marginating肝白细胞的选择性收获

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

右心导管插入术后天鹅管囊小贴中内皮细胞的提取

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

从小鼠肺中分离活髓细胞和上皮细胞群

从小鼠肺中分离活髓细胞和上皮细胞群

从小鼠肺中分离活髓细胞和上皮细胞群

软脑膜淋巴内皮细胞的收获和原代培养