我们感谢博士。亚历克斯格尔巴德和威廉Overwijk的流体力学基因转移的建议。这项工作是由美国国立卫生研究院（AI098099，西南），MD安德森癌症中心的表观遗传学，MD安德森中心的炎症和癌症（SSW），和RE鲍勃史密斯教育基金（HSL）的资金支持。

机构的批准必须提前对本文描述的所有实验操作涉及的小鼠获得。这些技术包括利用C57BL6小鼠的骨髓祖细胞的分离，IFNAR - / -小鼠为受体的造血祖细胞的过继转移和使用HGT的体内细胞因子过度表达。适当的住房及动物护理也必须由研究者或机构提供。此外，可能需要在流体动力学的基因转移（ 即重组DNA批准）中使用的质粒的机构批准。这里所描述的研究被批准的机构动物护理和使用委员会在UT MD安德森癌症中心。 1。水动力基因转移（HGT） 这一步应该提前2天个CDP的过继转移进行诱导循环FLT3L直流扩建体内 5。公关epare至少5受体小鼠/组。 <ol><li>实现高效的HGT，收件人IFNAR的血管- / -小鼠（CD45.2 +）应通过暴露小鼠的加热灯5-10分钟进行散瞳。 </li><li>鼠标放置在一个阻挡装置和消毒其尾部，用70％的乙醇。 </li><li>注射5微克的质粒的编码FLT3L于2ml无菌PBS到尾静脉用27号针头。对于控制队列，如上经尾静脉注射5微克空载体（PORF）的。 </li><li>监测小鼠15-30分钟，以确保有尾静脉注射无不良影响。返回小鼠房屋公用设施专用2天。 </li></ol> 2。从小鼠骨髓分离造血祖细胞的这一步应该HGT术后第2天进行。使用同类系CD45.1 +小鼠骨髓祖细胞的来源过继转移到受体IFNAR - /- 动物（CD45.2 +）。在这个协议中，同源品种需要区分供体和受体衍生的DC，以及避免免疫介导的消耗由于MHC错配。约10-20小鼠将被要求为转移实验提供足够数量的造血祖细胞（10 5个细胞/受体小鼠）。 <ol><li>用CO 2窒息和颈椎脱位安乐死10同源CD45.1 +小鼠。 </li><li>将每个胎体上的夹层托盘和消毒的腹部和腿部用70％乙醇。 </li><li>做一个切口在中间腹部和切断通过从腹部皮肤每条腿，切割皮肤下腿的长度。 </li><li>轻轻地从每个支腿去除皮肤和切割并从胎体去除腿在使用锋利的剪刀髋关节。 </li><li>小心地从每个支腿的用锋利的刀片的股骨和胫骨取出肌肉。 </li><li>清除股骨和胫骨用锋利的解剖刀的两端，并放置骨头中含有的完全RPMI培养皿（RMPI用10％热灭活的胎牛血清，1％青霉素/链霉素，1mM丙酮酸钠和50μM的β-巯基乙醇） 。 </li><li>准备含1ml完全RPMI中，连接到27号针头的注射器。 </li><li>插入27号针头插入股骨或胫骨的一端。通过轻轻地注入完全RPMI到骨头从股骨或胫骨冲洗骨髓。确保骨髓细胞正在从骨骼中移除，这可以通过在视觉上检测到出现浑浊排出介质来实现。 </li><li>重复冲洗每个股骨和胫骨3倍，以彻底去除骨髓细胞。 </li><li>通过轻轻地在培养皿上下吹打细胞3到5倍制备单细胞悬浮液。 </li><li>通过一个40微米的细胞过滤网的骨髓细胞，将所述细胞渗出到一个新的文化视角除去碎片E盘。扰乱，通过用无菌注射器活塞年底温柔的压力出现在过滤器的任何细胞团块。 </li><li>裂解红细胞（RBC）中存在的总的骨髓细胞悬浮液与商业的RBC裂解缓冲液根据制造商的说明进行操作。使用2毫升红细胞裂解buffer/4-6×10 7个细胞（细胞通常从一个鼠标）和5分钟，在室温温育时间。 </li><li>通过离心制粒细胞在500×g离心4分钟，轻轻地吸出培养基，再悬浮在10ml FACS缓冲液（1×PBS +2毫摩尔EDTA +1％FBS）中，造粒的细胞通过离心分离，并轻轻抽吸洗涤洗涤骨髓细胞缓冲区。 </li><li>重复洗涤，如在步骤2.13中所述，共洗涤2次。 </li></ol> 3。荧光激活细胞分选术（FACS）来隔离个CDP 这一步需要一个初始阴性选择过程访问MidiMACS细胞分离和MACS LD柱，以及在FACS机器具有至少3激光器用荧光标记的抗体染色后，以净化多色祖子集。 <ol><li>在步骤2.14第二次洗涤后，计数骨髓细胞。如在步骤2.13中所述通过离心沉淀细胞，重悬在FACS缓冲液中，以达到每30微升FACS缓冲液中4×10 7个细胞的终浓度。 </li><li>以制备细胞用于FACS，谱系阴性细胞首先通过在使用磁性珠柱色谱法从骨髓混合物除去谱系阳性细胞负选择富集技术。对于阴性选择过程中，添加1微克每下列商业大鼠抗小鼠抗体（ABS）能识别造血谱系标志物组成：CD3，CD19，CD11c的，CD11b的，和TER-119腹肌。的绝对值应当在一个容积为2微升/抗体对30微升细胞悬浮在FACS缓冲液被加入。 </li><li>孵育的细胞悬浮液在4℃下进行30分钟。以下的温育后，如在步骤2.13中所述两次，用10ml的FACS缓冲液洗涤细胞。 </li><li>重悬细胞，达到4×10 7 cells/40微升FACS缓冲液中的最终浓度。加入20微升的山羊抗大鼠IgG每份40微升细胞悬浮液的磁微珠。轻轻混匀孵育4℃，30分钟。如在步骤2.13中所述，用10ml的FACS缓冲液洗涤细胞。 </li><li>悬浮高达10 8骨髓细胞于500μlFACS缓冲液。 </li><li>加载MACS LD柱上按照制造商的说明，一MidiMACS细胞分离和预先清洗柱用2ml FACS缓冲液。 </li><li>适用的骨髓细胞悬浮于列，装载高达5×10 8个细胞在2.5ml FACS缓冲液中。确保收集管（15毫升锥形管）放置在柱下。洗柱3次2毫升流式细胞仪缓冲/清水冲洗，并收集穿过柱子，将富集的细胞谱系阴性细胞。计数细胞在材料中洗脱（洗通）从柱中。 </li><li>以通过FACS进行阳性选择的造血祖细胞亚群，染色细胞用下面的荧光共轭ABS：IL-7R（太平洋蓝），FLT3（PE）的Sca-1（PE.Cy7），CD115（APC）中，c-kit的（APC.Cy7）ABS，加上针对CD3，CD19，CD11c的，CD11b的，F4/80和TER119（所有标有PERCP经Cy5.5）标记的血统绝对的混合物。使用0.5-1微升各抗体在100微升的总体积。孵育细胞悬浮液在4℃下20-30分钟。 </li><li>在2-3×10 7细胞/ ml的浓度，如步骤2.13，重悬在FACS缓冲液中所示洗涤细胞。通过一个35微米的细胞过滤帽筒筛选细胞去除细胞团块，这最后一步是至关重要的，以避免堵塞FACS机器。 </li><li>将细胞悬浮在FACS管上流式细胞仪舞台和排序个CDP（林-的c-kit 罗 CD115 +的Flt3 +）对依据指定的标记谱的。收集在15ml试管含有5ml的完全RPMI中纯化细胞。 </li><li>记录分选的细胞的绝对数量在FACS运行结束。在无菌PBS在步骤2.13，重悬表示过继转移实验通过离心沉淀细胞。 </li></ol> 4。个CDP的过继转移这一步通常是在动物设施所在的受体小鼠被收容。取决于FACS机器的位置，这可涉及将纯化的祖细胞群的运输进入动物设施之前的过继转移。祖细胞悬浮液应保持无菌和输送在冰上。 <ol><li>通过在100微升无菌PBS中，总体积稀释10 5个CDP纯化制备细胞悬浮液的注射。把细胞悬浮在连接到27号针头的注射器抽出。 </li><li>揭露收件人IFNAR - / - </su对&gt;小鼠（CD45.2 +）以加热灯5-10分钟，通过尾静脉以实现高效喷射。 </li><li>鼠标放置在一个阻挡装置和消毒其尾部，用70％的乙醇。 </li><li>注入10 5 FACS纯化的在100μlPBS个CDP到尾静脉用27号针头。 </li><li>监测小鼠15-30分钟，以确保有尾静脉注射无不良影响。返回小鼠的住房设施。 </li></ol> 5。 PP的PDC和测量隔离金额<ol><li>在7-10天以下的过继转移，安乐死的受体小鼠。轻轻地剥离开鼠标和揭露肠。 </li><li>删除整个肠道，并将其放置在PBS浸湿的纸巾。沿着收集用细镊子和剪刀的小肠壁所有可见的PP。 C57BL6和IFNAR - / -小鼠通常有5-10 PP /鼠标，这是从发现孤立淋巴滤泡不同沿小肠18。请注意，该PPS往往结构不同，即使在同一个鼠标，所以仔细确保所有的PP被识别和解剖。 </li><li>放置的PP在含有PBS的培养皿中，并使用镊子除去粪便。重复此步骤两次清洁的PP。 </li><li>消化的PP与1毫克/毫升胶原酶中于50毫升10毫升1X Hank氏平衡盐溶液（HBSS）四烧瓶中，在剧烈搅拌1小时，在37℃下</li><li>通过过滤将消化的PP在40微米的细胞过滤和强制细胞的顶部隔间，用无菌注射器活塞。收集的PP细胞悬液15毫升锥形管。 </li><li>通过离心分离，在500 xg离心5分钟，并重新悬浮于6ml 37％的Percoll溶液（37％的Percoll在RPMI培养基）沉淀应变PP细胞。轻轻地放在6毫升70％的Percoll溶液（70％的Percoll在RPMI培养基）将细胞悬浮液的下面，以形成的Percoll梯度步骤。 </li><li>离心细胞20分钟一吨800 XG用离心机折断 。单核细胞会迁移到37/70％的界面。离心后，将单核细胞群应该由在界面区域小心移液被收集。沉淀收集的细胞通过离心分离并在50ml完全RPMI /洗涤的洗涤两次。大音量使用，以确保完全去除珀。 </li><li>染色收集PP细胞与下列抗体检测，从过继转移的祖细胞（CD45.1 +）或受体小鼠（CD45.2 +）小鼠中获得的pDCs：CD45.1（APC.Cy7），CD45.2（PE 。Cy7的），表面CD11c（太平洋蓝），细胞CD11b（PERCP经Cy5.5），B220（APC）的Siglec-H（PE）和PDCA-1（FITC）ABS。流式细胞仪PP细胞的分析步骤3.9和3.10所示进行流动。识别的pDCs由他们的CD11c + CD11b的- B220 +的Siglec-H + PDCA-1 +表型。 ％的pDCs x可：绝对的pDC数目可以由下式确定TAL PP细胞数= PP pDCs细胞。 </li></ol>

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本文所描述的过继转移技术评估个CDP到PP的pDC群在受体小鼠中有缺陷的聚丙烯的pDCs的贡献（ 例如IFNAR - / -小鼠）。在以后的实验中，这将是重要的，以评估其他祖亚群的电位在产生的PP的pDCs，特别是聚丙烯的pDCs是否从的Flt3 +的CLPs派生。这个问题是显著，因为目前还不清楚为什么PP pDCs细胞具有独特的敏感，适用于PP累计5 IFNAR-STAT1信号，而且如果PP的pDCs遵循类似的发?...

在这里，我们展示了一个协议，以评估共同树突状细胞祖细胞（个CDP）是否能够在浆细胞树突状细胞（PDC）人口淋巴集结（PP）引起。使用这种方法的总体目标是评估的pDC的淋巴集结（PP pDCs细胞）的发育调控。的原因，这是很重要的是，聚丙烯的pDCs从其他组织，包括骨髓，血液和脾脏中找到的pDCs不同的，因此，目前还不清楚PP的pDC和其他的pDC群是否发育和/或功能上相关的。具体来说，pDCs细胞被广泛称为是主要的I型干扰素（IFN）生产者造血系统中，响应Toll样受体7和9（TLR7 / 9）快速干扰素分泌1-3刺激。然而，PP的pDCs是缺乏生产型干扰素响应TLR激动剂刺激4,5。此外，聚丙烯的pDCs也从发现在骨髓和脾脏中的pDCs不同要求从类型信号I干扰素（IFN）受体（IFNAR1）或干扰素信号分子STAT1为他们的发展和/或积累5。这些数据表明，不同的监管机制，控制PP的pDCs与其他器官pDCs细胞（ 如骨髓，脾）5的可能性。 导致这一方法的发展的基本原理是基于在理解树突状细胞（DC）的生物学的最新进展。大多数，如果不是全部，DC亚群从表达该FMS-样酪氨酸激酶3受体（的Flt3）6-10造血祖细胞派生，但是，直流发展并不局限于经典的髓系和淋巴途径。例如，FLT3 +普通髓系祖细胞（中医师，林- IL-7R -的Sca-1 -的c-kit + CD34 +细胞FcγRLO / - ）来个CDP（林-的c-kit 罗 CD115 +的Flt3 +）引起，whic率进一步分化成PDC和传统的DC中心（CDC）9,10。相比之下，FLT3 +普通淋巴祖细胞（的CLP，林- IL-7R +的Sca-1 LO的c-kit LO），主要发展为pDCs细胞11。因此，以前的研究表明的pDCs产生下FLT3L的调节至少2个不同的造血祖细胞群体，虽然典型的分析已被限制在骨髓，脾脏和/或血液的pDC亚群。因此，生成聚丙烯的pDCs的祖人口（S）所需的调查。了解PP pDCs细胞的起源将揭示他们是否与其他的pDC人群有着共同的发展途径光线，或者他们这一代人在PP中使用不同的机制。 本文所述的方法的独特优点是使用，显示严重不足的PP pDCs细胞为受体的造血祖细胞的过继转移小鼠。小鼠基因抽动缺失该基因编码的IFNAR1（IFNAR - / -小鼠）或STAT1（STAT1 - / - ）揭示了PP的pDCs 5一个惊人的消耗。因此，这些菌株提供一种聚丙烯的pDCs被还原的环境，允许继转移研究，在缺少有效的细胞消融制度如致死照射来进行。这里提出的方法的另外一个优势是利用流体力学基因转移（HGT）来刺激FLT3L升高的循环量。这提供了以诱导FLT3L 体内 ，相对于注射重组蛋白的具成本效益的方法。大量的研究，包括那些在我们的实验室中，聘用HGT诱导细胞因子的量在各种实验条件5,12,13。 劳动和精确的免疫功能区议会的划分是在免疫学的主要兴趣。特别是，pDCs细胞是口服耐受和全身抗病毒的重要介质反应，但它们也似乎有助于自身免疫和癌症14-17的发展和持续性。本文所述的协议将允许调节聚丙烯的pDCs的发育机制更加充分的探讨。此外，这种方法可能会允许研究，以评估PP pDC的功能，并且可以扩展到理解中的PP其他免疫人群的调节和功能。

<table border="0" cellpadding="0" cellspacing="0" width="867">
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		<col />
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">C57BL/6J</td>
			<td style="width:201px;">JAX</td>
			<td style="width:177px;">664</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">B6.SJL</td>
			<td style="width:201px;">JAX</td>
			<td style="width:177px;">2014</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RPMI</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:177px;">11875-093</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">15 ml conical tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352095</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">50 ml conical tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352070</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile surgical tweezers</td>
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			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile small pair scissors</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile large pair scissors</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">40 &#956;m cell strainer</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352340</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">35 &#956;m cell strainer cap tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352235</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RBC lysing buffer</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:177px;">R7757</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:260px;">FACS buffer</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;">PBS, 2 mM EDTA, 1% FCS, filter sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Percoll</td>
			<td style="width:201px;">GE Healthcare</td>
			<td style="width:177px;">17089102</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">10XHBSS</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:177px;">H4641</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Collagenase IV</td>
			<td style="width:201px;">Worthington</td>
			<td style="width:177px;">LS004188</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Goat ant-rat IgG microbeads</td>
			<td style="width:201px;">Milteyni Biotec</td>
			<td style="width:177px;">130-048-501</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">LD column</td>
			<td style="width:201px;">Milteyni Biotec</td>
			<td style="width:177px;">130-042-901</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-D3</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">555273</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD19</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553783</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD11b</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553308</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD11c</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553799</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-Ter119</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553671</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD3 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0031</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD19 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0193</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11b (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0112</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11c (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0114</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-F4/80 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-4801</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Ter119 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-5921</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Sca-1 (PE.Cy7)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">25-5981</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD115 (APC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">17-1152</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-c-kit (APC.Cy7)&#160;</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">47-1171</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-IL-7R (Pacific Blue)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">48-1271</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Flt3 (PE)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">12-1351</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD45.1 (APC.Cy7)</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">560579</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD45.2 (PE.Cy7)</td>
			<td style="width:201px;">Biolegend</td>
			<td style="width:177px;">109830</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11c (Pacific Blue)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">48-0114</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-B220 (APC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">25-0452</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Siglec-H (PE)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">12-0333</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-PDCA-1 (FITC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">Nov-72</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cell sorter</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;"></td>
			<td style="width:229px;">e.g. BD Fortessa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Heat lamp</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Mouse restrainer</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1 ml syringes</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td style="width:177px;">309602</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">27&#189; G needles (sterile)</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td style="width:177px;">305109</td>
			<td style="width:229px;"></td>
		</tr>
	</tbody>
</table>

assessing the development of murine plasmacytoid dendritic cells in peyer's patches using adoptive transfer of hematopoietic progenitors

该协议详细介绍了分析纯化造血祖细胞在肠道淋巴集结（PP）的产生浆细胞树突状细胞（PDC）的能力的方法。常见的树突状细胞前体细胞（个CDP，林-的c-kit 罗 CD115 +的Flt3 +）从C57BL6小鼠的骨髓中纯化，通过FACS，并转移到缺乏一个显著的pDC群中的PP受体小鼠，在这种情况下，IFNAR - / -小鼠用作转移接收者。在某些小鼠中，树突状细胞生长因子Flt3配体（FLT3L）的过度表达到代管个CDP转移之前执行，使用FLT3L编码质粒流体力学基因转移（HGT）。 FLT3L过度扩张DC群转自（或内源性的）造血祖细胞起源。在祖转让后7-10天，即产生过继转移的祖细胞的pDCs从受体细胞进行区分的CD45标志物表达，与转个CDP是CD45.1 +和受助人CD45.2 + pDCs细胞的基础。转个CDP的贡献到PDC人口PP和向FLT3L反应能力是由从受体小鼠的PP单细胞悬液流式细胞仪检测。此方法可用于测试其他祖种群是否能够产生PP的pDCs的。此外，该方法可用于检查被预测影响的pDC发育中的PP，通过将前体亚群与推定的发育因子和/或一个适当的击倒，敲除或过表达由通过HGT操纵循环细胞因子是因子的作用。此方法还可以使聚丙烯的pDCs如何影响在其他的PP免疫亚群的频率或功能分析。这种方法的特点是采用IFNAR的- / -小鼠，这表明严重枯竭PP的pDC相对于野生型动物，从而allowiPP的pDCs在没有从致命的辐射影响因子纳克重建。

我们的结果表明，该门控策略个CDP的分离从小鼠骨髓细胞（ 图1），如在协议2和图3详细说明。个CDP包括总骨髓细胞的约0.1％，和大约4-6×10 4个CDP可以从一个小鼠中分离出来。一旦过继转移，个CDP分化为pDCs细胞和CDC 10。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51189/51189fig1highres.jpg" src="/files/ftp_upload/51189/51189fig1.j...

Watch this Scientific Journal Video about Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors at JoVE.com

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

这个协议描述的实验程序来评估浆细胞样树突细胞的Peyer氏从共同的树突状细胞前体细胞的补丁的分化，使用涉及FACS介导的细胞分离，水动力的基因转移，并在淋巴集结免疫子集流分析技术。

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

assessing-development-murine-plasmacytoid-dendritic-cells-peyer-s

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JoVE Journal

Immunology and Infection

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

11.1K 次观看.  The University of Texas MD Anderson Cancer Center. 这个协议描述的实验程序来评估浆细胞样树突细胞的Peyer氏从共同的树突状细胞前体细胞的补丁的分化，使用涉及FACS介导的细胞分离，水动力的基因转移，并在淋巴集结免疫子集流分析技术。 

视频: Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

免疫与感染

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...