We would like to thank Marilaine Fournier for her comments on the manuscript and Jos&#233;e Tessier for technical assistance. C57BL/6-Tg (OT-I)-RAG1&lt;tmMom&gt; #4175 were obtained through the NIAID Exchange Program, NIH. Support for this research is provided by a grant from the SickKids Foundation and CIHR-IHDCYN (NI15-002), an operating grant from the CIHR-III (MOP-142254), and start-up funds from the FRQS (&#201;tablissement de jeunes chercheurs) and H&#244;pital Maisonneuve-Rosemont Foundation to HJM. HJM is a junior 1 scholar of the FRQS, a CIHR New Investigator (MSH-141967), and a Cole Foundation Early Career Transition award recipient.

HOPITAL迈松内夫 - 罗斯芒特 - 适用于所有的动物研究协议是由动物护理委员会在该中心德RECHERCHE批准。 1.收获小鼠胸腺胸腺片的制备及单细胞悬浮液<ol><li>安乐死用CO 2鼠标颈椎脱位。 </li><li>在层流罩，针鼠标腹侧到夹层板。喷用70％乙醇鼠标。用纱布涂抹，以防止乙醇进入胸腔和破坏组织取出多余的酒精。 </li><li>解除在用一对镊子的胸骨的底部的皮肤，使通过皮肤切口。向上延伸的皮肤切口到每个前肢。 </li><li>使胸骨到膜片从肋骨分离的基础上额外削减。然后切向锁骨肋骨的两侧。翻转左肋过朝头部露出胸腔。的两个叶胸腺趴在心脏的顶部。 </li><li>使用镊子，显微解剖剪除去周围的胸腺裂片结缔组织和/或尖锐弯曲的剪刀。使用一对精尖弯钳从下方或通过剩余的结缔组织解除个别叶。 注意:不要直接掌握胸腺。 </li><li>放置在胸腺叶含PBS 15毫升的锥形管，直到需要预留冰上。 </li></ol> 2.琼脂糖嵌入和胸腺叶Vibratome切片<ol><li>制备4％的琼脂糖溶液为通过将2 G低低熔点琼脂糖在50ml无菌PBS中并在低设置微波直到琼脂糖完全溶解嵌入。覆盖在在55℃的水浴中铝箔和地点烧瓶直到准备使用。 </li><li>在组织培养罩，制备含有10％胎牛血清的完全RPMI-1640培养基（FBS），4毫摩尔L-谷氨酰胺，1×青霉素/链霉素和10μM的2-巯基乙醇。 </li><li>每孔加1.5毫升完全RPMI-1640培养基以6孔细胞培养板，并放置在每个一细胞培养插管井。在37℃下，直到需要拨该板在培养箱中。 </li><li>在冰桶准备与水和冰的泥泞冰水浴中。 </li><li>小心取出周围用细尖镊子各胸腺叶所有剩余的结缔组织。而胸腺瓣在PBS淹没在组织培养皿中，后转移到一个组织用PBS擦拭浸泡，或在解剖显微镜下做到这一点。 </li><li>允许琼脂糖冷却到低于40℃，当该烧瓶只是温暖的触感，以避免过热的组织。琼脂糖倒入冷却的4％到组织模具中的约1厘米的高度。 </li><li>用一对镊子的胸腺叶仔细转移到纸巾擦拭和轻轻摇动它以干燥它不破坏组织。确保组织完全干燥，否则将切片中滑出琼脂糖。 </li><li>小心将叶放入琼脂糖和其定位或者水平（以增加每个切片的表面积）或垂直于模具的底部（以增加片的数量）。放置在冰水模具5-10分钟，以使琼脂糖固化。 </li><li>在这段时间内，通过安装在缓冲盘，装配在试样盘，并插入vibratome刀片准备切断用vibratome。放置在试样盘上的片实验室胶带。消毒用70％的乙醇清洗，组装工作区。 </li><li>一旦琼脂糖凝固，翻转模具并轻轻按下，在其中心发布琼脂糖嵌入叶。 </li><li>使用锋利的刀片在底部修剪多余的琼脂糖围绕叶留下〜2毫米琼脂糖两侧各约0.5厘米</li><li>固定每个琼脂糖块与组织胶的样品盘上的一块胶布的下降。多个块可以粘在磁带上。 </li><li>对齐vibratome刀片无线TH琼脂糖块的顶部。用无菌PBS填充缓冲区托盘直到刀片和琼脂糖块（多个）完全浸没。 </li><li>部琼脂糖包埋的组织，得到切片的厚度为400-500微米。的vibratome设置0.225毫米/秒的速度，100赫兹的频率，和5°角。 </li><li>使用弯曲铲收集胸腺切片放入含有无菌PBS组织培养板，因为他们被切断。丢弃第一个和最后切片。 </li><li>检查在放大4倍光显微镜下的切片。选择与完整胸腺组织和周围琼脂糖（ 图1A）的片。 </li><li>转移片，用枪头轻轻弯曲铲胸腺片滑入细胞培养插管步骤2.3准备的板块。每个刀片可容纳〜3片。确保片不互相接触或细胞培养插入墙壁。 </li><li>直到需要保持在37℃的板。 </li></ol><p类="jove_title"&gt; 3。胸腺细胞的制备叠加<ol><li>隔离胸腺裂片如第1中所述手动解离波瓣使使用填充用5ml无菌PBS含2％FBS的灭菌15毫升组织研磨机单细胞悬浮液。转移细胞到15毫升锥形管中。 </li><li>离心将细胞在545×g离心在4℃下5分钟。弃去上清液，在室温下悬浮细胞在1ml 1×ACK裂解缓冲液（0.15M的氯化铵 ，10毫KHCO 3，0.1mM的钠2 EDTA）中的3分钟以溶解红细胞。 </li><li>所述管填充到15ml含2％FBS的PBS中。离心将细胞在545×g离心在4℃下5分钟。 </li><li>悬浮细胞在10毫升含2％FBS的PBS中，并通过255微米的筛网过滤器传递的细胞。 </li><li>算使用血球细胞。离心在545×g离心5分钟将细胞在4℃下，并在一个精矿重悬在PBS中含有2％FBS的细胞沉淀每毫升1×10 7个细胞的通货膨胀。 </li><li>标记将细胞与如果不使用胸腺小鼠表达同类系的标记或基因编码荧光报道从片内源性的细胞区分，根据制造商的协议蜂窝染料如羧基琥珀酰亚胺酯（CFSE）。 </li><li>标记的细胞的最后一次洗涤后，重悬在完全RPMI-1640培养基将细胞沉淀以每15微升1-3×10 6个细胞。 </li></ol> 4.胸腺细胞叠加到胸腺片<ol><li>用移液管抽吸周围的刀片胸腺切片的任何液体。 注:请注意，以避免损坏琼脂糖。如果该琼脂糖已损坏，重叠胸腺细胞不会附着到片表面由于表面张力的损失。 </li><li>如果没有枪头，覆盖触摸片15微升第3节准备到每个胸腺切片胸腺细胞。 </li><li>孵育解放军德在37℃下2小时，以使细胞迁移进入胸腺切片。 </li><li> 2小时后，冲洗胸腺切片3次，用1毫升的PBS以除去尚未迁移进入组织过量重叠的胸腺细胞。 </li><li>根据实验在37℃下的板直到胸腺切片准备收获，典型地1-72小时。 </li></ol> 5.胸腺切片流式细胞仪的解离<ol><li>加入的PBS 150微升含有2％FBS的准备为每个​​切片一个离心管中。 </li><li>加入1毫升含2％FBS的与胸腺切片组织培养插入物的PBS并用弯曲刮铲轻轻搅动以从细胞培养插入物的表面分离的片。 </li><li>从使用弯曲抹刀插入到微量离心管转移的切片。 </li><li>手动扰乱使用离心管样品杵切片。添加的PBS 150微升含有2％FBS的至最终体积300微升。 </li><li>过滤通过40微米的过滤器中的解离组织到一个新的离心管中。将过滤的细胞准备被染色流式细胞仪分析40。 </li><li>将它们传递上的流式细胞仪，以确保所有的琼脂糖被除去，不仪堵塞流之前进行过滤的细胞第二次。 </li><li>获取关于一个流动细胞仪和如先前已描述40分析数据。 </li></ol>

The authors declare that they have no competing financial interests.

在这里，我们描述了胸腺切片并通过流式细胞术重叠预选择-MHC I类限制性TCR转基因的胸腺细胞的高效阳性和阴性选择的代表性结果的制备的协议。此系统已用于具有类似的成功支持从预选DP胸腺细胞32 MHC II型限制的CD4 + T细胞的阳性选择，并且，在激动剂抗原，阴性选择和胸腺T 章发展11,12的存在， 36,38,39,43,44。该协议可以被修改为研究抑制剂，定义的肽的?...

T细胞分化，通过在此期间，他们遇到几个检查点，以确保一个功能，自我耐受T细胞库1-3的产生胸腺一系列发育中间体。阳性选择促进与T细胞受体（TCR），能够识别的，具有低到中度亲和力，肽由皮质胸腺上皮细胞（CTEC）2,3-主要组织相容性复合体分子（MHC）呈现胸腺细胞的存活。阴性选择和调节性T（T REG）细胞发育有助于通过消除或转移强烈由MHC 2,4提出自身肽反应胸腺细胞的建立自身耐受。未成熟CD4 + CD8 +双阳性（DP）胸腺细胞表达了通过选择过程分化成成熟T细胞亚群的TCR，其中大部分是MHC I类限制性CD8 +细胞毒性或MHC II类限制性CD4 +辅助单阳性（SP）T细胞中，离开胸腺在次级淋巴器官1-3执行效应器功能之前。 添加到T细胞发育的复杂性是动态迁移并在整个基质细胞网络5-9显影胸腺细胞的细胞接触。这些基质细胞在胸腺细胞发展中发挥不同的作用，并在那里阳性和阴性选择发生10胸腺皮质和髓质区之间被差异分布。虽然正选择主要发生在皮质，有越来越多的证据认为DP胸腺细胞迁移到髓质和继续需要的TCR信号它们分化成成熟T细胞暗示髓质可提供必要的正选择和谱系完成附加的信号之前分化11,12。进一步，尽管表达专门髓质胸腺上皮细胞（MTEC）和本组织限制性抗原促进自身反应性胸腺13,14的缺失的情况下，阴性选择的相当大的比例发生在响应皮层到遍在表达自身肽树突呈现细胞15,16。因此，T细胞发育的准确的模型必须提供高度组织胸腺微环境，具有完整皮层和髓质区，便于胸腺细胞和基质细胞之间的相互作用，并支持胸腺细胞迁移，因为这些细胞经历阳性和阴性选择。 为了补充体外胸腺细胞分析作为研究阳性和阴性选择，一些在体外，原位的手段，并在 T细胞发育的体内模型已经开发17-22。它已经非常难以概括在体外的积极选择，但表达Notch配体干细胞群或基质细胞T细胞前体共培养，特别是OP9-DL1 / 4的细胞，具有支持T系的承诺和有限的正向选择的能力使其成为一个非常宝贵的体外模型研究T细胞发育23-25。本系统的限制，但是，包括这些细胞缺乏胸腺基质细胞上发现的唯一的肽加工机械和三维胸腺微环境的事实。 虽然在技术上更为繁琐，就地和胸腺选择的体内模型可以克服一些与体外系统的障碍。 Reaggregate胸腺器官培养（RTOC）包含定义的胸腺细胞和胸腺基质细胞18,26,27的混合物。这些胸腺上皮细胞reaggregates保持MHC I类和II表达并且可以支持DEVELOPME常规T细胞亚群的NT，但仍然缺乏定义的皮质和髓质结构。胎儿胸腺器官培养（FTOC）是T细胞发育的一个流行的模型，可以通过lymphodepleted胸腺叶或通过胸腺细胞注射悬滴文化的胸腺细胞被接种到lymphoreplete胸腺叶和支持CD4 +和CD8 + T的高效发展在细胞培养18,28-31时间。在胎儿胸腺瓣培养开始有mTECs的缺乏，但根据条件定义皮质和髓质结构可以随时间发展。一个重要的考虑是，该模型可优先支持胎儿对成人T细胞的发展。最后，在成年小鼠胸腺中定义的前体胸腺内注射在技术上具有挑战性，但显然提供了一个环境，以支持 T细胞发育体内 。这些原位 和体内模型是优秀的快工具Ø研究T细胞发育，其使用应在一个实验按实验基础上加以考虑。 胸腺片，然而，最近已成为一个通用的，互补的模型，研究胸腺选择就地与可能性，以适应独特的，复杂的，并且通常更高的吞吐量实验。胸腺片保持皮质和髓质区的完整性并提供基质细胞的框架，开发以及高效阳性和阴性选择11,32-39期间支持胸腺细胞迁移。添加顶上胸腺切片胸腺细胞亚群迁移到组织到相应的微环境利基34,37。的重叠胸腺细胞可以从胸腺切片内源性细胞经由同类标记或荧光标记来区分，并可以在培养物中维持数天。胸腺器官切片文化，可以用来研究的各个方面T细胞发展，包括胸腺选择，胸腺细胞行为（迁移和细胞相互作用），和胸腺细胞定位，等等。给生成每只小鼠〜20胸腺切片的能力，实验的可扩展性通常比在胸腺选择的原位模型其他更大。虽然胸腺切片的制备需要专门的设备，如vibratome，并且由于通过细胞死亡随着时间的推移细胞的损失和缺乏包封膜被限制在培养胸腺片的使用寿命，胸腺片提供了一个极好的模型对于一个成熟的胸腺微环境胸腺细胞同步群体的胸腺选择的分析。在这里，我们描述了胸腺片（包括收获的胸腺，胸腺裂片的琼脂糖嵌入和嵌入的组织的vibratome切片），隔离和胸腺细胞的重叠，并解离为流式细胞仪分析胸腺切片的制备方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>VT1000S&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>NuSieve GTG Agarose</td>
			<td>Lonza</td>
			<td>50080</td>
			<td>Low melting temperature agarose</td>
		</tr>
		<tr>
			<td>Embedding Mold (Truncated - T12)</td>
			<td>Polyciences</td>
			<td>18986</td>
			<td>22mm x 22mm square, truncated to 12mm x 12mm</td>
		</tr>
		<tr>
			<td>Double Edge Prep Blades</td>
			<td>Personna</td>
			<td>74-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Adhesive</td>
			<td>3M&#160;</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4 &#181;m Cell Culture Inserts&#160;</td>
			<td>BD Falcon</td>
			<td>353090</td>
			<td>Of several brands tested, these maintained the cells atop the slices the best</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher</td>
			<td>21600-010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 with L-glutamine</td>
			<td>Wisent</td>
			<td>350-000-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Wisent</td>
			<td>080-110</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>L-Glutamine, 200mM</td>
			<td>Wisent</td>
			<td>609-065-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin, 100X</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Alfa Aesar</td>
			<td>A15890</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Tenbroeck Tissue Grinders</td>
			<td>Wheaton</td>
			<td>357426</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Filter</td>
			<td>Component Supply</td>
			<td>U-CMN-255</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tube Sample Pestle</td>
			<td>Bel-Art</td>
			<td>F19922-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Nylon Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Inox Tip</td>
			<td>Dumont&#160;</td>
			<td>RS-5047</td>
			<td>Fine tip curved forceps, size .17&#160;X .10mm&#160;</td>
		</tr>
		<tr>
			<td>Micro Forceps</td>
			<td>Dumont&#160;</td>
			<td>RS-5090&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and applications of organotypic thymic slice cultures

在产生了功能性的，自我耐受T细胞库的产生一种独特的和高度有组织的胸腺微胸腺选择前进， 在体外模型来研究T系承诺和发展已经提供了有价值的见解这一过程。然而，这些系统缺乏必需的T细胞发育的完整三维胸腺环境，因此， 在体内胸腺选择的不完全近似。一些涉及到建模T细胞发育的挑战可以通过使用原位模型提供了一个完整的胸腺微环境完全支持显影性T细胞的胸腺选择来克服。胸腺器官切片培养补充现有的原位技术。胸腺切片保存胸腺皮质和髓质区的完整性，并提供了一​​个平台，研究一个限定的发育阶段的或内源T C的重叠胸腺细胞发育一个成熟的胸腺微环境中厄尔。鉴于生成每只小鼠〜20片的能力，胸腺切片进行高通量实验的可扩展性方面呈现出独特的优势。此外，在生成胸腺切片和电位从不同遗传背景叠加不同胸腺子集或其它的细胞群相对容易提高该方法的多功能性。这里，我们描述的胸腺片，隔离和胸腺细胞的叠加，以及流式细胞仪分析胸腺片分离制备协议。这个系统还可以适于研究非常规T细胞的发育以及可视胸腺细胞迁移，胸腺间质细胞相互作用，并通过双光子显微镜与胸腺选择相关联的TCR信号。

T细胞发育的不同方面，如阳性和阴性选择胸腺切片支持分析。对于成功的实验中，胸腺切片的质量是至关重要的。 因此，应研究胸腺切片以确保胸腺组织的完整性和周围胸腺切片琼脂糖是完好（ 图1A）。当琼脂糖损坏导致在迁移进入组织胸腺细胞的数目显著减少表面张力就会受到损害。因此，具有组织损伤或刻痕的适应症胸腺片/眼泪在琼脂糖?...

Watch this Scientific Journal Video about Preparation and Applications of Organotypic Thymic Slice Cultures at JoVE.com

Preparation and Applications of Organotypic Thymic Slice Cultures

我们描述的是，在用流式细胞仪组合，可以被用来建模显影T细胞的阳性和阴性选择胸腺切片的制备方法。胸腺片也可以适用于胸腺细胞迁移，本地化的原位分析，并通过免疫荧光和双光子显微镜信令。

制备及胸腺器官切片文化中的应用

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

preparation-and-applications-of-organotypic-thymic-slice-cultures

Research

JoVE Journal

Immunology and Infection

8.0K 次观看.  Hôpital Maisonneuve-Rosemont. 我们描述的是，在用流式细胞仪组合，可以被用来建模显影T细胞的阳性和阴性选择胸腺切片的制备方法。胸腺片也可以适用于胸腺细胞迁移，本地化的原位分析，并通过免疫荧光和双光子显微镜信令。 

视频: Preparation and Applications of Organotypic Thymic Slice Cultures

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

胸腺淋巴瘤的T细胞系的推导 ATM - / -</sup P53 - / -</sup鼠标

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

科研

免疫与感染

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

在野生鸟类禽流感病毒快速诊断:使用便携式的RRT - PCR和在外地的冻干试剂

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

制备肿瘤抗原负载成熟树突状细胞免疫治疗

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

促进FACS纯化的胸腺上皮细胞的3-D聚合，EAK 16-II / EAKIIH6自组装凝胶

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

制备及胸腺器官切片文化中的应用

摘要

探索更多视频

此视频中的章节

胸腺淋巴瘤的T细胞系的推导 ATM^{- / - P53^{- / -鼠标}}

在野生鸟类禽流感病毒快速诊断:使用便携式的RRT - PCR和在外地的冻干试剂

从核衣壳的病毒DNA的制备

从小学的人角质形成细胞器官筏文化的代

考试流式细胞仪检测胸腺阳性和阴性选择

制备肿瘤抗原负载成熟树突状细胞免疫治疗

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

隔离和不同的老年小鼠胸腺移植的移植。

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

促进FACS纯化的胸腺上皮细胞的3-D聚合，EAK 16-II / EAKIIH6自组装凝胶

制备及胸腺器官切片文化中的应用

摘要

探索更多视频

此视频中的章节

胸腺淋巴瘤的T细胞系的推导 ATM - / - P53 - / -鼠标

在野生鸟类禽流感病毒快速诊断:使用便携式的RRT - PCR和在外地的冻干试剂

从核衣壳的病毒DNA的制备

从小学的人角质形成细胞器官筏文化的代

考试流式细胞仪检测胸腺阳性和阴性选择

制备肿瘤抗原负载成熟树突状细胞免疫治疗

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

隔离和不同的老年小鼠胸腺移植的移植。

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

促进FACS纯化的胸腺上皮细胞的3-D聚合，EAK 16-II / EAKIIH6自组装凝胶

胸腺淋巴瘤的T细胞系的推导 ATM^{- / - P53^{- / -鼠标}}