这项工作是由美国国立卫生研究院的PBS（R21 AI095484）的支持。我们感谢健保四聚体核心设施的CD1d四聚体。

1，准备工具，培养基和试剂<ol><li>工具:准备试剂在无菌条件下并在层流罩中使用。 </li><li>制备1升的RPMI组织培养基中，添加胎牛血清的5％，2.1克碳酸氢盐，将10毫升谷氨酸100倍，10毫克的HEPES，1毫克丙酮酸钠和10毫升的100×青霉素/链霉素。 注意:推荐的组织培养基，RPMI，是可互换使用的DMEM。 </li><li>制备5％胶原酶的"D"（5毫升胶原酶的"D"来9毫升组织培养基）。 </li><li>作出的Percoll溶液在三种不同浓度:100％，80％和40％。以下浓度计算1样品。保持溶液在室温下。 <ol><li>珀解决方案:添加9毫升稀释珀（来自原液）;加入1 ml的PBS的10倍。 </li><li>珀解决方案:添加8毫升珀的100％;加入2毫升的PBS 1X的。 </li><li>珀的解决方案:加入4 mL的Percoll 80％;加入4毫升的PBS 1X的。 </li></ol></li><li>使1升荧光激活细胞分选（FACS）缓冲液;加入5 ml的10％BSA（0.1％BSA）中，加入1ml 10％的叠氮化钠（0.1％），将2ml EDTA剂，使高达1000毫升的1×PBS。 </li></ol> 2，准备肾消解<ol><li>已经获取任何必要的机构批准，牺牲和抽血的小鼠与标准方法并按照现行的法规和照顾动物的指引。 <ol><li>麻醉小鼠用戊巴比妥（0.07毫克/千克）。 </li><li>继续放血。 </li><li>鼠标放置在上手术台仰卧位置。 </li><li>喷雾用70％乙醇的腹部区域。 </li></ol></li><li>加入10毫升的胶原酶D溶液的培养皿中。 </li><li>取出并从每个鼠标拆封两个肾脏。 <ol><li>通过腹部皮肤和腹膜从胸骨到做一个正中切口打开abdomimal腔耻骨。 </li><li>通过横向移动肠道内侧面暴露左肾。 </li><li>仔细分离左侧胶囊镊子。 注意:该胶囊显示为围绕肾透明层和部分地覆盖由肾周围脂肪组织。 </li><li>用手术剪刀切断蒂部血管取出左肾。 </li><li>重复同样的程序右肾。 注:右肾更颅，更接近中线（花梗较短）。 </li></ol></li><li>将肾入培养皿中。 </li><li>使用常规金属成形刀片切肾组织切成小块（1-2毫米直径），然后将片变成为在孵化30-45分钟的胶原酶溶液在37℃（ 图1）。 </li></ol> 3，从肾脏消化制备肾单核细胞（KMNC）的<ol><li>获取肾脏的细胞悬浮液消化用在25毫升的组织培养介质的过滤器（70微米），重悬和混合机械破坏的组织。 </li><li>离心机在400×g离心10分钟，在4℃下沉淀的细胞悬浮液。 </li><li>重新悬浮于4ml的40％的Percoll溶液沉淀，轻轻地覆盖到加入4ml 80％的Percoll溶液与转印塑料吸管，非常缓慢和小心。这将导致在两个阶段通过一个半透明的层显然分开。顶部将是对应于脂质黄色层。 </li><li>在制动切断模式离心机离心以1,500 xg离心30分钟，在室温下进行。 </li><li>通过抽吸去除黄而厚的顶层（约1-2毫升）。 </li><li>收集仅轻微发白的半透明的两相的界面层用移液管。该悬浮液很小心转移到15毫升锥形管中含2ml组织培养基，然后添加13毫升介质，使总体积15毫升。 </li><li>离心机在400×g离心10分钟，在4℃下</li><li>重悬样品在1ml的培养基中。 </li><li>使用台盼蓝排斥于血细胞计数器计数KMNC的数量和表达的结果作为KMNC的每肾脏数每毫升。 </li><li>洗如上一次。 </li></ol> 4，造血隔离（CD45 +）的KMNC细胞（步骤一） <ol><li>重悬细胞高达10 7到90微升缓冲液中。 </li><li>加10微升CD45微珠并孵育15分钟，在4℃下</li><li>加入1 ml的运行缓冲液，使反应停止。离心机在400×g下在500μl的缓冲液中4℃，重悬10分钟。 </li><li>通过放置在磁铁准备磁列和冲洗用3毫升缓冲液中。 </li><li>通过磁性柱通过将细胞悬浮液，并收集通过柱子的未标记细胞。 </li><li>用3毫升银行经营的洗细胞三次样缓冲液。然后收集污水。 </li><li>从分离器和吸管5毫升电泳缓冲液到列中删除该列，并立即冲洗掉标记部分。这部分包含了CD45 +细胞。计数细胞。 </li><li>计算不同的T细胞亚群的绝对数为肾脏由阳性细胞的百分比通过流式细胞术测定（ 图2）相乘KMNC的总数。 </li></ol> 5，隔离的DN性T细胞的CD45 +编写的由负选择（步骤二） <ol><li>添加2.5微升（0.5毫克/毫升，1.25微克）下列各生物素化抗体的:抗-CD4，抗CD8，抗Fc受体（CD16/32），抗-II类MHC（IA b）所示，抗CD1d的PBS-57四聚体，每10 7造血细胞。 </li><li>孵育细胞在冷室中30分钟。 </li><li>加入1 ml的抗生物素微珠。 </li><li>孵育在冷室中30分钟。 </li><li>将管在T他磁体和进行消耗。 </li><li>计数存活的细胞用血细胞计数器确定DN T细胞的负分数的总数。 </li><li>检查的DN T细胞的纯度流式细胞仪由以下这门控策略:第一栅CD45 +，第二栅极TCRαβ+。排除的CD1d +细胞。情节的CD4 +与CD8 +识别DN T细胞（ 图3）。 </li><li>通过在纯度确定的百分比相乘的总数。 注:在超过90％的纯DN T细胞的人口使用该协议的结果。 </li></ol>

<ol>
	<li>Zhang, Z. X., Yang, L., Young, K. J., DuTemple, B., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+previously+unknown+antigen-specific+regulatory+T+cell+and+its+mechanism+of+suppression.">Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression.</a> Nat Med. 6, 782-789 (2000).</li><li>Ford, M. S., Young, K. J., Zhang, Z., Ohashi, P. S., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+regulatory+function+of+lymphoproliferative+double+negative+T+cells+in+vitro+and+in+vivo.">The immune regulatory function of lymphoproliferative double negative T cells in vitro and in vivo.</a> J Exp Med. 196, 261-267 (2002).</li><li>Ford McIntyre, M. S., Young, K. J., Gao, J., Joe, B., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+in+vivo+trogocytosis+as+a+mechanism+of+double+negative+regulatory+T+cell-mediated+antigen-specific+suppression.">Cutting edge: in vivo trogocytosis as a mechanism of double negative regulatory T cell-mediated antigen-specific suppression.</a> J Immunol. 181, 2271-2275 (2008).</li><li>Young, K. J., Yang, L., Phillips, M. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-lymphocyte+infusion+induces+transplantation+tolerance+by+activating+systemic+and+graft-infiltrating+double-negative+regulatory+T+cells.">Donor-lymphocyte infusion induces transplantation tolerance by activating systemic and graft-infiltrating double-negative regulatory T cells.</a> Blood. 100, 3408-3414 (2002).</li><li>Chen, W., Ford, M. S., Young, K. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+in+vitro-generated+DN+T+regulatory+cells+induces+permanent+cardiac+allograft+survival+in+mice.">Infusion of in vitro-generated DN T regulatory cells induces permanent cardiac allograft survival in mice.</a> Transplant Proc. 35, 2479-2480 (2003).</li><li>Young, K. J., DuTemple, B., Phillips, M. 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作者什么都没有透露。

有一个在DN T细胞增加的兴趣，因为他们被牵连在不同的病理情况，如自身免疫性疾病，癌症，移植耐受和肾脏，包括急性肾损伤（AKI）的原发疾病，肾小球肾炎8，13。因此，有必要更好地理解和描述的DN T细胞的病理生理功能。然而，目前仍缺乏这些细胞的功能相比，CD4和CD8 T细胞的了解。一个主要的原因是DN T细胞在次级淋巴器官的缺乏，因此难以取得足够的细胞在体外分析和过继...

A correction was made to: <a href="http://www.jove.com/video/51192/isolation-of-double-negative-t-cells-from-the-kidney">Isolation of Double Negative &#945;&#946; T Cells from the Kidney</a>.
There was an error with an author's name. The author's given name had a typo, this was corrected to:
Samatha Bandapelle
instead of:
Samantha Bandapelle

A correction was made to: Isolation of Double Negative &#945;&#946; T Cells from the Kidney.

Erratum: Isolation of Double Negative &#945;&#946; T Cells from the Kidney

周边αβTCR+ CD3 + CD4 - CD8 -双阴性（DN）T细胞被分成具有不同的表型和功能1-4各种子集。 DN T细胞知之甚少，但越来越多地被牵连在不同的疾病模型4-6病理免疫反应。 DN T细胞，是在各种免疫和自身免疫应答的调节和异体移植耐受的调制日益牵连了独特的免疫细胞类型。4-6，9它们是罕见的外周血和次级淋巴器官（脾和淋巴结）。然而，它们的正常肾和肠上皮10-12主要居民。很少有人知道他们在稳定状态功能7在肾和病理条件下，如肾transpl相关的急性肾损伤（AKI）下蚂蚁。 因为不同的免疫细胞在调节免疫反应，包括alloresponses，限定每个游戏者的角色的错综复杂的角色是在理解alloresponses和设计新疗法至关重要。给出的显著号码存在于生理和不同疾病状态下的肾DN T细胞，DN的T细胞很可能在移植受者起到在小鼠和人类调节免疫和自身免疫应答的关键作用，并且alloresponses。积累虽然仍散落证据牵连的DN T细胞在致病性和免疫功能，但它是知之甚少，为什么，以及他们如何表现出特定的有害或抑制功能和环境如何影响他们。 由于它们的低丰度在肾脏，分离的改进的方法是必要的。 目前还没有标准协议，用于从T DN T细胞的分离他非淋巴组织。我们的协议描述了一种新颖的方法，用于从肾脏DN T细胞的隔离;然而，也可用于各种非淋巴组织的方法。

<table border="0" cellpadding="0" cellspacing="0" width="1172">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Laminar flow hood&#160;</td>
			<td style="width:257px;">Baker Company, Inc</td>
			<td style="width:276px;"></td>
			<td style="width:200px;">Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hemocytometer</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:276px;">63514-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Atmosphere-controlled incubator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td style="width:200px;">&#160;(37&#176;C with 5% CO2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Microscope&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Analytical flow cytometer</td>
			<td style="width:257px;">LSR II&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">Name of the reagent</td>
			<td style="width:257px;">Company&#160;</td>
			<td style="width:276px;">Catalog&#160; Number</td>
			<td style="width:200px;">Comments&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">RPMI 1640</td>
			<td style="width:257px;">Media Tech</td>
			<td style="width:276px;">10-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Collagenase D</td>
			<td style="width:257px;">Roche</td>
			<td style="width:276px;">11088858001</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Percoll&#160;</td>
			<td style="width:257px;">GE Healthcare</td>
			<td style="width:276px;">&#160;17-0891-01</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fetal bovine serum&#160;</td>
			<td>Corning Cellgro</td>
			<td>35-011-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">0.1% sodium azide (NaN3)</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">S2002</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Buffer phosphate buffered saline (PBS)</td>
			<td style="width:257px;">Corning Cellgro</td>
			<td>21-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">PBS 10X</td>
			<td>Mediatech</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">EDTA buffer&#160;</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">E1161</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">LS columns&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">CD45+ microbeads</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">6.78E-51</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Biotin microbeads&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-090-485</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; PerCP&#160;</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td style="width:276px;">G1397</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; APC-Cy7</td>
			<td style="width:257px;">Biolegend&#160;&#160;</td>
			<td style="width:276px;">103116</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-TCR Pacific Blue&#160; (ab-chain, clone: H57-597)</td>
			<td style="width:257px;">Invitrogen</td>
			<td style="width:276px;">HM3628</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 PE</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>12-0043</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 FITC</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>8011-0087</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 biotinylated (GK1.5)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553728</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 biotinylated&#160; (clone 53-6.7)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553029</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Fc receptor (CD16/32) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553143</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-MHC class II (I-A d) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553609</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD1d PBS-57 tetramer</td>
			<td style="width:257px;">NHI tetramer&#160;</td>
			<td style="width:276px;">lote # 15621-PBS57</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">AutoMACS Running Buffer-&#160; MACS Separation Buffer&#160;</td>
			<td style="width:257px;">MIlteny I Biotec</td>
			<td style="width:276px;">130-091-221</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">C57BL/6J mice</td>
			<td>Jackson Labs</td>
			<td style="width:276px;">000664</td>
			<td>Kidneys - Lymph Nodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri dish&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:276px;">356517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">70mc filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plunger&#160;</td>
			<td style="width:257px;"></td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeneMate Tubes 50 ml</td>
			<td>Bioexpress</td>
			<td>C-3394-3</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">GeneMate Tubes 15 ml</td>
			<td>Bioexpress</td>
			<td style="width:276px;">C-3394-1</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri Dish&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:276px;">S33580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plate 24 wells&#160;</td>
			<td style="width:257px;">BD Biosciences</td>
			<td style="width:276px;">354723</td>
			<td style="width:200px;"></td>
		</tr>
	</tbody>
</table>

isolation of double negative &#945;&#946; t cells from the kidney

目前还没有标准协议，用于DN T细胞从非淋巴组织的隔离，尽管它们在不同的免疫应答日益报道的参与。 DN T细胞是有牵连的调 ​​节免疫和自身免疫应答和耐受allotransplants 1-6独特的免疫细胞类型。然而DN T细胞，罕见的外周血和次级淋巴器官（脾脏和淋巴结），但都在正常肾脏的主要居民。很少有人知道他们的病理生理功能7，由于其在外围缺乏。最近，我们在稳定状态，并在缺血再灌注损伤中描述的肾脏8这部分人口的综合表型和功能分析。的DN T细胞功能分析将通过开发一个协议，用于从肾脏的隔离大大提高。 在这里，我们描述了一种新的协议，允许isolatioÑ​​高纯度AB的CD4 + CD8 + T细胞和DN T细胞的小鼠肾脏。简单地说，我们用胶原酶消化肾组织，并通过密度梯度分离肾单核细胞（KMNC）。这是由两个步骤，从KMNC丰富的造血T细胞从3％至70％。第一步骤包括一个正选择使用CD45阳性分离试剂盒的造血细胞。在第二步骤中，DN T细胞被除去使用CD4，CD8，和MHC II类的单克隆抗体和CD1d的α-GalCer的四聚体的非期望的细胞的负分离。这个策略导致了90％以上的纯DN T细胞群。表面染色用上述抗体随后通过FACS分析用于确定纯度。

野生型C57BL / 6（B6）肾包含每个肾约1.5-2.1×10 6单核细胞。约小于10％是造血CD45 +细胞。对于肾脏单核细胞（KMNC）的制备中，肾脏切成小块， 如图1后跟消化，用5％的胶原酶。 其次是通过执行一个密度梯度离心来收集KMNC层（图2，左图）。 KMNC然后用CD45 +的磁性微球作为在第4（步骤I）所述受到正选择。列结合的细胞，然后通过FACS洗脱?...

Watch this Scientific Journal Video about Isolation of Double Negative ÃŽÂ±ÃŽÂ² T Cells from the Kidney at JoVE.com

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

DNabT细胞是罕见的外周T细胞之间;然而，他们是丰富的在某些非淋巴组织。从非淋巴组织中分离的DN T细胞的难度阻碍了其功能的分析尽管越来越多的认可的病理生理意义。我们描述了一种新的协议，用于从小鼠肾脏高度纯化的DN T细胞的隔离。

从肾脏双重否定αβ性T细胞的分离

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

isolation-of-double-negative-t-cells-from-the-kidney

Research

JoVE Journal

Immunology and Infection

12.1K 次观看.  Johns Hopkins University School of Medicine.  DNabT细胞是罕见的外周T细胞之间;然而，他们是丰富的在某些非淋巴组织。从非淋巴组织中分离的DN T细胞的难度阻碍了其功能的分析尽管越来越多的认可的病理生理意义。我们描述了一种新的协议，用于从小鼠肾脏高度纯化的DN T细胞的隔离。 

视频: Isolation of Double Negative αβ T Cells from the Kidney

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

科研

免疫与感染

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

从大鼠睾丸支持细胞的分离和管周细胞

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...