我们感谢史蒂夫McBryant和杰夫汉森鼠标操作的帮助与超速离心和帕蒂凯泽帮助。授予5R01NS056379 - 02在美国国立卫生研究院，美国国家神经疾病和中风研究所资助的这项工作。

<html> 1。净化和标签朊病毒棒该协议是改编自一个以前出版了18本 <ol><li>在900毫升的冰冷的均质缓冲朊病毒感染的脑组织匀浆100克（HB，1X PBS含320毫米蔗糖，氯化钠150毫米和4毫米EDTA）1分钟，最大速度在商业搅拌机，然后在冰上2分钟。重复三次。 </li><li>在3000 XG 10分钟和4 ° C离心匀浆删除，并保存在冰上清。重新悬浮颗粒在1L HB和重复步骤1.1和1.2。 </li><li>池上清液和离心100000 XG，0 ° C 60分钟弃上清。 </li><li>重新悬浮颗粒在100毫升的1X三缓冲液（10毫米的Tris - HCl，氯化钠0.1毫米和1毫米EDTA）含2％Triton X - 100的的（TBST）。布拉德福德或类似的实验估计蛋白浓度调整至5毫克/毫升在TBST。冰浴30分钟。 </li><li> 100000 XG，0 ° C离心30分钟弃去上清液，沉淀用50毫升TBST 100毫升的TBS。重新悬浮颗粒在100 mL 1X PBS中含1％sarcosyl和蛋白酶抑制剂的鸡尾酒和120分钟的搅拌，在37 ° C。 </li><li> 900毫升蔗糖320毫米和60分钟的离心样品10万，10 ° C XG层弃上清，重新悬浮颗粒在2.3M氯​​化钠10毫升，5％sarcosyl。 </li><li>超声在70％的最大功率在1分钟间隔冰样本5 × 40秒。 </li><li>分装1毫升样品到Eppendorf管和离心13000 XG，摄氏4度15分钟。洗净用TBS和存储颗粒的两倍@ -70 ° C或共轭荧光步骤1.9。 </li><li>共轭1管纯化的朊蛋白棒，使用1小瓶DyLight 649荧光，根据制造商的协议。 </li><li>交易所DyLight标签对PBS缓冲液透析或通过离心过滤列。商店在20μL分装在-70℃避光，直到准备使用。 </li></ol> 2。朊病毒接种和细胞恢复本节包括朊病毒接种腹腔和腹膜，纵隔，肠系膜淋巴结和脾脏细胞的恢复。 <ol><li>稀释荧光朊病毒棒的PBS 1:50之前使用。用拇指和食指背侧颈部皮毛颈背鼠标，轻轻转动鼠标，面对你和约束小指尾。旋后鼠标，提升头以上的后腿躯干和他略有倒挂。 </li><li>使用30G的胰岛素注射器，注入100μL荧光朊病毒棒中线和骶髂关节前1-2厘米的外侧1厘米。插入针经皮肤1厘米的内侧45 °。使用1:50稀释的荧光珠或PBS单独作为对照。分析前的时间分配，孵育小鼠。 </li><li>安乐死小鼠根据经批准的动物护理和使用委员会的协议。使用12 ml注射器和25G针进入腹膜腔注入10毫升的PBS。轻轻摇动胴体纵向一分钟。在冰上使用相同的注射器和16G针到15 mL锥形管和储备，而胴体的不断清扫腹膜恢复细胞悬液。 </li><li>用70％乙醇彻底湿润的胴体的腹侧方。电梯在正下方用钳胸腔中线的皮肤，并通过皮肤用剪刀切一个小切口。有开始，通过在两个方向上的头部和尾部的皮肤3-5厘米长的切口，然后从每端两个2厘米的切口从中线横向。剥开针皮肤揭示腹腔和胸腔。小心地穿过薄的半透明膜周围的腹膜，反映胸廓的揭示纵隔。 </li><li>查找和删除2-4纵隔淋巴结肿大，稍背和横向相邻胸腺，靠近气管的腹内侧相邻臂动脉，。查找并删除六个肠系膜淋巴结，嵌在柔软的白色肠系膜组织肠子锚后腹壁。查找并删除脾，位于中线外侧和背在左侧腹部的肠子长，暗红色的器官。储备淋巴结清扫术是在1 mL的RPMI 1640培养基在冰上，直到完成。 </li><li>浸渍，并通过40微米到3毫升的RPMI网状过滤器在一个塑料的Petri菜用1 mL注射器的柱塞按淋巴结。冲洗过滤器和一个额外的2毫升的RPMI 5 mL细胞悬液转移到15 mL锥形管。额外的5毫升的RPMI冲洗培养皿转移到同一管内。 250 XG离心5分钟，弃上清，重新悬浮细胞沉淀在1 mL流式细胞仪缓冲液（1 × PBS，1％牛血清白蛋白和10 mM EDTA）和转移到1.5 mL Eppendorf管。 </li></ol><html> ove_title“&gt; 3。细胞染色和流式细胞仪下面是一个典型的染色协议确定对良好的免疫细胞表面标记的荧光抗体的免疫细胞亚群。使用前，所有的抗体流式细胞缓冲液稀释成。 <ol><li>到Eppendorf管中的细胞和分装10 6细胞计数。离心机细胞（见2.7）和流式细胞仪用1 mL缓冲液洗颗粒2X。 </li><li>孵育细胞在冰上20分钟，在100μL2毫微克/毫升的鼠抗鼠CD16/32以阻断内源性的Fc受体。沉淀和流式细胞仪缓冲液洗细胞。 </li><li>加入100μL1ng /毫升适当的荧光antibod（Y）IES每个样品在冰上培育一个小时。添加100μL流式细胞仪缓冲单独控制细胞。沉淀和洗涤细胞与流式细胞仪缓冲两次。 </li><li>在1毫升ACK缓冲液（150毫米，1毫米KHCO 3和0.1毫米EDTA的NH 4 CL）1分钟，以溶解红细胞冰孵育细胞，重新挂起。沉淀和流式细胞仪缓冲液洗细胞。 1毫升流式细胞仪缓冲液重悬细胞。 </li><li>收集细胞，使用流式细胞仪多色检测和分析的能力，通常与405，488和633 nm激发激光器和五至十年的荧光发射探测器的数据。 </li></ol> 4。代表性的成果： 从原油中分离纯化棒朊病毒，朊病毒感染的脑组织匀浆（ 图1）洗涤剂溶解和E2的麋鹿脑组织匀浆离心（ 泳道1和2）极大地丰富了朊病毒棒（比较泳道1和3），这也是蛋白酶K耐（4车道）。有趣的是，未消化的纯化朊病毒棒显示一个glycoform轮廓惊人地相似，消化E2的脑组织匀浆，表明朊病毒戏剧性的浓缩。相同处理的正常脑组织匀浆产生任何的PK抗性的PrP C（5和6） 。 流的特定细胞群的流式细胞仪分析表明，抗原提呈细胞两小时回家腹膜和留住荧光珠和朊病毒棒，大大增加了PBS注射控制荧光（ 图 2）证明。从上述背景荧光（ 图2A - G）的显示控制PBS治疗腹膜收获细胞，而细胞珠（ 图2H - N）和朊病毒（ 图2O - U）接种小鼠显示显着的荧光，特别是在单核细胞（图2I和 P），树突状细胞（图2K和 R）和巨噬细胞（图2L和 S），和一些中性粒细胞（图2J和Q）和B 细胞较少 （图2T）。珠和朊病毒轴承单核细胞，树突状细胞和巨噬细胞中也发现了两小时后接种（ 图2AD和AK，自动对焦和AM和AG和AN，分别），而很少或根本没有的PrP CWD的轴承中性粒细胞的纵隔淋巴结，B或T细胞被发现（ 图2AE和AL，AH和AG和AI和AP，分别）。我们没有发现珠脾或肠系膜淋巴结（数据未显示）或朊病毒棒。在TOTO，这些数据表明，免疫细胞的朊病毒交通非常类似于其他微粒抗原。 <table border="1"><tbody><tr><td> 试剂 </td><td> 荧光 </td><td> 激发光（NM） </td><td> 峰值发射（纳米） </td></tr><tr><td>朊病毒棒</td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> 1微米的小珠</td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> 抗体 </td><td></td><td></td><td></td></tr><tr><td> CD11b的</td><td> eFluor 450 </td><td> 405 </td><td> 450 </td></tr><tr><td></td><td>藻红蛋白- Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td>的CD11c </td><td> R -藻红蛋白</td><td> 488 </td><td> 575 </td></tr><tr><td></td><td>藻红蛋白- Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> Ly6C </td><td> Fluoroisothiocyante </td><td> 488 </td><td> 518 </td></tr><tr><td> Ly6G </td><td> R -藻红蛋白</td><td> 488 </td><td> 575 </td></tr><tr><td> CD21 </td><td> DyLight 488 </td><td> 488 </td><td> 518 </td></tr><tr><td> B220 </td><td>变藻蓝蛋白，Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> CD3 </td><td> R -藻红蛋白</td><td> 488 </td><td> 575 </td></tr></tbody></table>荧光试剂的光谱特性“&gt;表1 。 <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="图1" /> 图1。从被感染的脑组织匀浆的朊病 ​​毒棒净化我们从感染CWD的鹿（E2，1和2车道）作为起始原料，从中我们纯化汇总朊病毒棒（泳道3和4）10％的原油脑组织匀浆。原油和纯化的材料表明：蛋白酶K处理的特性阻抗（1 - 4车道），而正常脑组织匀浆不（5和6车道）。 Kd以印迹左侧分子量标记显示。波黑，脑组织匀浆。 <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="图2" /> 图2。流式细胞仪分析免疫细胞贩运朊病毒从腹膜，纵隔淋巴结肿大，两小时后接种 。PBS（板公司的V - AB），荧光珠（HN和AC - AI）或朊病毒杆（OU和AJ - AP ）注入小鼠腹腔灌洗液（非盟）或纵隔淋巴结肿大（V型AP），两小时后收获细胞腹膜。图中的第一列显示细胞治疗的小鼠与PBS（面板A和V），荧光珠（面板H和AC）和朊病毒棒（板O和AJ）。绘制荧光细胞（红色或绿色的点）和总细胞数（灰色点）显示的相对规模（前向散射），粒度（侧散射）和总活细胞，荧光的比例。粒细胞特征的细胞大量保留了荧光珠和棒。细胞是还沾有抗免疫细胞表面标志物，并为连云港的Ly6C +单核细胞门（板B，I，P，W，公元和AK），Ly6c -的CD11c的细胞CD11b + Ly6G +中性粒细胞（C，J，Q，X，自动曝光的抗体和AL），Ly6G - Ly6C -细胞CD11b +的CD11c +树突状细胞（ð，K，R，Y，自动对焦和下午），Ly6G - Ly6C -的CD11c的细胞CD11b +巨噬细胞，（发送，大号，小号，ž，公司和AN），CD3 + - B220 + CD21 + B细胞（F，M，T，AA，AH和AO）和CD21 - B220 - CD3 + T细胞（G，N，U，AB，AI和AP）。单核细胞，树突状细胞和巨噬细胞保留在腹腔内（板我和P，K和R和L和S，分别）荧光珠和朊病毒棒和运输纵隔淋巴结（AD和AK面板，自动对焦和AM和AG分别），表现出类似的摄取这两种微粒和贩运。中性粒细胞保留了大量的珠（J）和较少的杆（Q）的，但没有提供他们淋巴结（AE和AL）。 B细胞保留，并运送一些棒（T和AO），但几乎没有珠（M和AH）。 T细胞被贩卖既不珠也不棒（N，U，AI和AP）。

<ol>
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在这里，我们展示了一个标记和跟踪朊病毒的协议 ，在体内，大大方便了监控周边朊病毒感染的早期事件。该协议大大提高了监测朊病毒吸收过去的尝试标签前的高浓缩铀朊病毒接种 9体外和 体内 3,12明确区分内源性的PrP彗星。由于传染性朊病毒PrP的彗星共享相同的主要的氨基酸序列，产生朊蛋白的特异性抗体一直是个问题。我们已经执行了?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CWD-infected elk brain</td>
<td>&nbsp;</td>
<td>Private elk farm in Colorado</td>
<td>&nbsp;</td>
<td>Use any non-human prion-infected brain</td>
</tr>
<tr>
<td>Blender</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>6694-015</td>
<td>Use any commercial blender</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>SS34 rotor</td>
<td>Use any centrifuge /rotor that can reach 3000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>50.2 Ti rotor</td>
<td>Use any ultracentrifuge /rotor that can reach 100,000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Bradford Reagent</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B6916</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>&nbsp;</td>
<tr>
<td>Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>MP4000X</td>
<td>Use any horn or probe sonicator set to ~70% max power</td>
</tr>
<tr>
<td>DyLight antibody Labeling kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>53050</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>55430R</td>
<td>Use any refrigerated microcentrifuge that can achieve 13,000x g</td>
</tr>
<tr>
<td>centrifugal filter columns</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>Microcon YM-100</td>
<td>Use any filter or dialysis membrane with 100 Kd molecular weight cutoff</td>
</tr>
<tr>
<td>8-40 week-old FVB mice</td>
<td>&nbsp;</td>
<td>Charles River</td>
<td>207</td>
<td>Use any inbred mouse strain</td>
</tr>
<tr>
<td>1 &mu;m red fluorescent beads</td>
<td>&nbsp;</td>
<td>Phosphorex</td>
<td>2307</td>
<td>Use any fluorescent bead &le; 10 &mu;m</td>
</tr>
<tr>
<td>RPMI 1640 medium </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>fluorescent antibodies</td>
<td>&nbsp;</td>
<td>BD pharmingen</td>
<td>Various</td>
<td>Use any fluorescent antibody appropriate for your application.</td>
</tr>
<tr>
<td>flow cytometer</td>
<td>&nbsp;</td>
<td>Dakocytomation</td>
<td>CyanADP</td>
<td>Use any flow cytometer capable of multicolor fluorescence detection</td>
</tr>		</tbody>
		</table>
		</div>

monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection

异常的形式存在的主机编码朊蛋白（PrPC）是蛋白酶耐，病理和传染性的朊病毒疾病，如慢性消耗病（CWD）鹿和羊痒病的特征。断言，这种不正常的构象构成的大部分或全部的传染性朊病毒，朊病毒假说。的免疫系统在外围朊病毒的发病机制中的早期事件中的作用已令人信服地证明 CWD和痒病1-3。在保留和复制朊病毒感染后4-6年初，在小鼠的基因和药理学研究发现补体系统的重要作用 。7-10树突状细胞在体外和体内研究也观察到朊病毒保留，尽管他们在贩卖的作用仍然不清楚11-16。巨噬细胞也同样被牵连在早期朊病毒的发病机制，但这些研究都集中在事件发生后感染3,11,17周。这些以前的研究也受到来自内源性的PrP C和传染性朊病毒之间的区别问题。在这里，我们描述了一个半定量的评估接种部位招募免疫细胞朊蛋白的吸收和贩运，不带偏见的方法。从被感染的脑组织匀浆洗涤剂溶解非聚合蛋白，通过蔗糖垫层离心纯化聚合朊病毒棒。聚丙烯酰胺凝胶电泳，考马斯亮蓝染色和免疫印迹证实颗粒分数高浓缩铀的朊病毒棒的回收。朊病毒棒荧光标记，然后注射到小鼠腹腔内。两个小时后测定腹腔灌洗液，脾，纵隔，肠系膜淋巴结的免疫细胞朊病毒杆保留和多色流量使用单核细胞，中性粒细胞，树突状细胞，巨噬细胞和B细胞和T细胞的标记流式细胞仪确定的细胞亚群。此试验允许首次直接监测的免疫收购细胞，感染后数小时内贩运朊病毒在体内。此实验也明确区分汇总朊病毒的传染性，从PrPC通常在宿主细胞中所表达的，可困难，并导致其他检测系统中的数据解释问题。可以适应其他接种途径（口服，静脉，intranervous和皮下，如）和抗原（共轭珠，细菌，病毒和寄生虫病原体和蛋白质，鸡蛋），以及该协议。

Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

在这里，我们描述了一种新的分析监测朊蛋白的吸收和免疫细胞的活动，通过净化后立即腹腔注射和荧光标记从被感染的大脑，然后监测其吸收和运动从注射部位和调解这些事件的细胞特征的材料汇总朊病毒棒。

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

monitoring-immune-cells-trafficking-fluorescent-prion-rods-hours

Research

JoVE Journal

Immunology and Infection

11.4K 次观看.  Colorado State University. 在这里，我们描述了一种新的分析监测朊蛋白的吸收和免疫细胞的活动，通过净化后立即腹腔注射和荧光标记从被感染的大脑，然后监测其吸收和运动从注射部位和调解这些事件的细胞特征的材料汇总朊病毒棒。

视频: Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

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

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

心脏功能免疫细胞的分离

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

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

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

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

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

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

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

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...