医学真菌学真菌免疫学（SFB，JMB，JK，AW） - 这项工作已经由MRC和阿伯丁大学（MRC中心医学真菌学）和威康信托基金会战略奖支持。特别感谢的是从Chloe的资金支持。 TMH是由国立卫生研究院（NIH，代码RO1 093808）和纪念斯隆·凯特林癌症中心的资助是由美国国立卫生研究院授予P30 CA008748支撑。另外，TMH是传染病由宝来惠康基金支持的发病机制的调查。我们要感谢仔显微镜和组织学设施对他们的帮助和支持。

用于获得中性粒细胞和外周血单核细胞（PBMC）的协议是基于以前出版的方法9。使用来自健康志愿者的血液样本已通过阿伯丁大学的人类研究伦理委员会。开始之前，确保所有的伦理审批到位从健康志愿者和/或病人抽血所描述实验的目的。在冰上保持在可能情况下，以增加他们的生存细胞。 1. 烟曲霉文化条件<ol><li>如所描述的10制备葡萄糖基本琼脂培养基上。 <ol><li>调整媒体使用1M NaOH将pH为6.5并高压釜在120℃下20分钟。 </li><li>使其冷却至65℃。 </li><li>在无菌流罩的工作，倒入30毫升冷却介质成T75烧瓶中并允许与静止烧瓶的颈部冷却上的25毫升吸移管至create琼脂斜面。 </li></ol></li><li>条纹列于琼脂DSRED Af293- 烟曲霉菌株，在37℃+/- 5％CO 2孵育7天。两个烧瓶将在5mL的磷酸盐缓冲盐水（PBS）预生物素化，得到大约10 9个分生孢子。 </li></ol> 2. 烟曲霉标签和制备注意:当首次执行该测定中，制备亲本Af293- 烟曲霉菌株。采取两种菌株的样品中的微量离心管中并且如下所述仅标记每个菌株中的一个。这将允许一个有控制的分生孢子没有颜色和分生孢子用单一颜色，无论是红色荧光蛋白或AF633，测试设置和设备。 <ol><li>通过用30mL PBS + 0.05％吐温-80中浸渍培养收获烟曲霉分生孢子。通过40微米的细胞过滤到50mL管过滤所得悬浮液，以除去菌丝片段。 </li><li> Centrifuge在805×g离心10分钟，除去上清液，并在20毫升无菌PBS洗一次。在洗涤步骤中从相同菌株的两个板池分生孢子。 </li><li>在洗涤步骤之后，重新悬浮于5mL PBS的颗粒和转印分生孢子悬浮液的1ml等份到微量离心管中。 1mL的悬浮液各菌株的通常足以用于活细胞成像。包裹在箔和存储剩余的等分试样在4℃下。 </li><li>在9300×g下在室温下10分钟，并小心地分生孢子悬浮液的等分试样离心除去上清液。重悬沉淀分生孢子在1mL的0.05M的NaHCO 3 pH值8.3。 </li><li>通过在500μL的DMSO重构25毫克生物素制备10毫克生物素/ 200μL二甲亚砜（DMSO）储备液。添加10μL生物素/ DMSO原液到微量离心管中，在盖铝箔，并培育2小时上在4℃下的摇杆。 <ol><li>分装的生物素/ DMSO股票搜索解决方案的剩余部分n和在-20℃下，在随后的实验中使用的冻结。 </li></ol></li><li>离心10分钟，在9300×g下，小心地除去上清。重悬在1mL的100mM的Tris-HCl的pH 8.0的分生孢子沉淀1个小时以失活自由浮动生物素。 </li><li>离心10分钟，在9300×g下，小心地除去上清。用1mL无菌PBS洗涤沉淀两次，重悬沉淀在1mL的PBS中。 </li><li>溶解1毫克链霉亲和AF633的0.5毫升的PBS使一个2mg / mL的储备溶液。加入2 mg / mL的链霉亲和AF633的10μL每1mL分生孢子悬浮液的孵育在室温下在铝箔覆盖摇臂40分钟。其余的链霉亲和AF633可以等分被冻结在随后的实验中使用。 </li><li>离心10分钟，在9300×g下，小心地取出上清。重悬沉淀分生孢子在1mL的PBS中，并在1计数使用血球分生孢子:1,000稀释液（1:100，然后1:10）。调整分生孢子浓度3.6×10 6个 / mL的用CO 2独立媒体，在4℃下在箔和存储包裹。 注:现在的分生孢子都标有AF633，并将作为FLARE分生孢子被称为。 </li><li>可选:如果需要与肿分生孢子刺激，计数分生孢子（步骤2.9）后，稀释分生孢子至7.2×10 6个 / mL，在酵母氮基（YNB）培养基中，并在高压灭菌50加入500μL分生孢子悬浮液至4.5毫升YNB培养基姆·埃伦迈尔瓶中。覆盖并放置在烧瓶6小时振荡培养箱（37℃200rpm下）。 <ol><li>所述介质转移到15毫升管。离心机中以805×g于室温下10分钟，并在PBS中洗涤一次。再次离心，重悬沉淀在1毫升CO 2独立介质，给予3.6×10 6个分生孢子/ ml。 注:分生孢子经常聚集他们变得肿胀进行准确的计算困难后，建议用C来计算ounted数字静止使用分生孢子。 </li></ol></li></ol> 3.人体中性粒细胞和单核细胞的分离<ol><li>绘制20毫升的来自健康志愿者的静脉血液到含有乙二胺四乙酸（EDTA）2个10毫升血管中。用于分开每个供体，倒入20毫升血液中的50mL管中，用15mL PBS稀释。 </li><li>下垫的血液用15ml淋巴细胞分离溶液（密度1.077克/毫升）用注射器和针铁的。放置针在管的底部，轻轻迫使淋巴细胞分离溶液进行。离心机中以630×g离心20分钟，不制动和低加速。 </li><li>准备PBS，在冰上冷却。 注意:步骤3.4和3.5必须同时遵循以产生的单核细胞（3.4）和嗜中性粒细胞（3.5）。 </li><li>使用pastette，收获PBMC层。这是在黄色血清（上）和透明淋巴细胞分离溶液（低级）层之间的中心的层，和转移到新的50ml管中。 <ol><li>装满PBMC管高达50毫升用冷PBS和离心机中以582×g离心10分钟。去除上清，每20毫升的供体血液最初使用在1mL的PBS中用冷PBS重悬洗两次。 </li><li>使1:10稀释液通过将细胞悬浮液于160μLPBS中和台盼蓝的20μL的20μL计数。计数和血球细胞。 </li><li>通过加入26毫升热灭活的胎牛血清（FCS）和2.1毫升的0.5M的EDTA至500mL HBSS的制备缓冲溶液。离心将细胞在515×g离心10分钟，并再悬浮于每1×10 7细胞缓冲溶液40μL。 </li><li>细胞悬液转移到15毫升管中并每1×10 7细胞添加CD14微珠的10μL，拌匀，并在4℃下孵育15分钟，确保所述管混合，每5分钟。 </li><li>通过加入每1×10 7个细胞和CEN缓冲溶液1ml洗涤细胞trifuge在515×g离心10分钟。 </li><li>吸去上清液完全，重悬到1×10个8个细胞中的缓冲溶液500微升。 </li><li>根据制造商的说明放置每个样品1列上的磁分离器。首先用缓冲液500μL洗涤柱一次。 </li><li>通过该柱吸取细胞悬浮液的500μL，等待柱干燥，然后通过用缓冲溶液重复该三次洗涤。 </li><li>将新的15毫升管中的列的下面，并采取柱切断电磁分离器。然后冲洗细胞出列入用1mL的缓冲溶液的试管中。 </li><li>加入4毫升缓冲溶液和离心机在515×g离心10分钟，然后在1mL的Roswell Park Memorial研究所（RPMI）培养基重悬。 </li><li>使1:10稀释液通过将细胞悬浮液于160μLPBS中和台盼蓝的20μL的20μL计数。计数和细胞血球。 </li><li>调节细胞浓度为6×10 5 / mL的用RPMI和在基于玻璃35毫米成像菜种子200μL。在37℃用5％CO 2孵育过夜。 </li><li>第二天，在成像之前，取出RPMI并添加CO 2独立介质的200μL。 注意:这些单核细胞也可以分化成成像之前各种巨噬细胞子集。如果这是要探索的技术，一个很好的起点是由Ohradanova-Repic 等人最近的纸张。 1。 </li></ol></li><li>收获PBMC层后，除去所有的血清和大部分淋巴细胞分离溶液，但要小心，不要除去上冲粒料，因为这将包含大多数的嗜中性粒细胞的顶部上的小的白色带。 <ol><li>通过添加83克NH 4的Cl和在1升无菌水10克KHCO 3的制备10个的低渗裂解缓冲液贮液。保存在4°C。 </li><li>这个稀释10倍的股票用无菌水，并把它添加到管。然后，小心地反转3次，并在冰上孵育15分钟。 </li><li>离心机中以394×g离心10分钟，并再悬浮在低渗裂解缓冲液在冰中10分钟。 </li><li>离心机394 XG，用冷PBS洗两次。重悬在1mL每个供体的CO 2独立培养基。 </li><li>使1:10稀释液通过将细胞悬浮液于160μLPBS中和台盼蓝的20μL的20μL计数。计数与血球细胞。 </li><li>用于流式细胞术，调至浓度为6×10 5 / mL和400μL细胞悬浮液添加到24孔板中。对于显微镜，添加相同的细胞悬浮液的200μL到基于玻璃35毫米成像盘中。 注:中性粒细胞成像或流式细胞术应立即开始，由于他们的倾向，如果没有刺激发生凋亡。如果这是不可能的嗜中性粒细胞应保持在冰并尽快用作可能的（在同一天之内）。 </li></ol></li></ol> 4.流式细胞仪<ol><li>添加1.2×10 6 / mL的FLARE分生孢子200μL到在24孔板中的细胞中，然后添加20μg/ ml的伏立康唑的100μL。添加RPMI的300μL，并且在37℃用5％CO 2孵育16小时。 加入伏立康唑，防止孢子萌发:注意。 </li><li>添加25μL1 mM的卡尔科弗卢尔白色原液向每个孔为25μm的终浓度，并在37℃用5％CO 2孵育10分钟。 </li><li>离心机中以582×g离心10分钟。除去上清液，并用PBS洗两次。 </li><li>收获贴壁细胞（单核细胞）中，用3mM的EDTA添加1毫升的PBS至每个孔，并在37℃用5％CO 2孵育10分钟。轻轻用移液管洗涤细胞从板上收集的细胞在管中。 </li><li>在FACS缓冲液洗一次（2％胎牛血清到PBS），然后在FACS缓冲液悬浮于FACS分析。 <ol><li>对于FACS分析，首先流式细胞仪调节流量的补偿参数，并设置使用单彩色补偿管正面门，包括分生孢子没有颜色:红色荧光蛋白+分生孢子，AF633 +分生孢子，并卡尔科弗卢尔白+分生孢子（如在4.2生成）。 </li><li>设置免费分生孢子门上了基于使用没有颜色的分生孢子侧向散射。通过排除免费分生孢子门上的前向和侧向散射设置单元门。 </li><li>通过记录10,000个事件分析样品管中。 注:用活分生孢子相关细胞将红色荧光蛋白+和AF633 +。与死亡有关的分生孢子细胞只会AF633 +。那些没有从事分生孢子会dsRed-和AF633-旁观者细胞。分生孢子，从事细胞群进一步分析了太平洋上的蓝色通道卡尔科弗卢尔白染色。已经内化分生孢子仍然存在细胞外将卡尔科弗卢尔白+，和分生孢子会卡尔科弗卢尔白色 - 。 </li></ol></li></ol> 5.活细胞显微视频注意:显微镜的选择将取决于什么是本地可用的，但在显微镜的设置将需要包括倒置的阶段，加热至37℃和适当的激发/发射过滤器的DsRed（五百六十一分之五百三十二nm）与AF633环境室（633纳米）。 FLARE分生孢子不与488 nm激光代替五百六十一分之五百三十二nm激光对红色荧光蛋白的工作。当与此第一次执行该实验中，使用控制分生孢子没有颜色和单色测试对于背景荧光和红色荧光蛋白和AF633信道之间的渗出通过。 <ol><li>打开之前的实验显微镜加热器，并允许足够的时间使环境控制腔室温至37℃。以稳定采取的腔室温度的时间将不同的显微镜设置而变化。 </li><li>打开显微镜和计算机，LO广告中的成像软件。安装在显微镜载物台的摄像培养皿中并调节焦距以找到人类嗜中性粒细胞或单核细胞。 </li><li>对于红色荧光蛋白和AF633，在采集设置设定的激光功率到10％和曝光时间为1秒。 </li><li>取出成像滑动，并添加100微升的3×10 6个 / mL的FLARE分生孢子悬浮液到适当的孔中（总体积300μL/孔）。录制FLARE分生孢子添加到菜的时间。 </li><li>返回滑动到舞台和调整的DsRed和AF633摄像机的灵敏度清楚地看到分生孢子但不是过度曝光图像。如果需要多点采集设置一个阶段点列表。 </li><li>开始成像时的所有点都在焦点中，通道被优化和循环时间和持续时间被设置。周期时间和成像持续时间取决于试验问题。的目标是保持周期时间尽可能地短（≤2分钟）1分钟在UPT的深入分析允许AKE动态。 </li></ol>

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Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes.</a> J Clin Invest. 69 (3), 617-631 (1982).</li></ol>

作者什么都没有透露。

该方法描述了一种新颖的体外方法，研究对烟曲霉的人原代巨噬细胞使用荧光曲霉记者（FLARE）分生孢子，活细胞成像的抗真菌活性和流式细胞术。先前的研究已经证明了无论是在鼠实验真菌感染体内 和抗真菌免疫6,7的体外评估使用FLARE分生孢子的附加值。组合FLARE分生孢子与此下一代活细胞成像技术允许一个在安装过程中在体外动态相互作用以测量单个分生孢子生存能力。 所描述的方法来研究它们的抗真菌反应的免疫细胞的某些细胞过程的具体作用和重要性的潜力。此外，从患特定患者吞噬细胞的抗真菌反应在他们的免疫系统中定义的单个组件的缺陷可以更详细地评估。很早就和初始抗真菌响应可以非常详细地在连续成像的6小时进行研究。这允许待检测甚至细微的差别，如巨噬细胞迁移的朝向真菌的速度，内化速率，杀真菌事件12，这将使用常规方法的吞噬作用是无法区分的动力学。 任何细胞类型都可以在本方法中使用;选择在此使用的细胞类型，因为这些吞噬细胞构成防御在人呼吸道的第一和第二线对烟曲霉 13。有趣的是，可以单核细胞之间和中性粒细胞是如何与休息和肿分生孢子和菌丝接合可以观察到非常清晰的差异。单核细胞几乎没有迁移到分生孢子，而嗜中性粒细胞显示更多的迁徙活动。附加荧光三月KERS如免疫细胞活死标记可以被包括在试验提供见解以下曲霉相互作用吞噬细胞的生存。该技术有趣潜在的未来应用包括不同的宿主细胞类型的共培养物中，抗真菌反应的后果（ 例如 ，在上皮单层，单核细胞衍生的树突状细胞与嗜中性粒细胞一起巨噬细胞），和反应性的实时测量以下用曲霉刺激氧的产生或嗜中性粒细胞的胞外陷阱的形成。 有几点需要注意使用该协议，最重要的是需要在实验室设置:具有倒置阶段的显微镜，环境室稳定在37℃，激发/发射过滤器的DsRed（五百六十一分之五百三十二nm）与AF633（ 633纳米）。显微镜，以保持细胞在聚焦和保留油浸（米时特别相关的能力ULTI点采集）应该被周期性地监测由于成像的持续时间长。对于杀菌活性的定量流式细胞仪设备是必需的。此外，适当的体制和伦理审批需要在地方与健康供者和患者的血液样本和基因操纵真菌工作。强烈建议使用新鲜的血液样本（在同一天使用），增加细胞活力和维护功能。理想情况下，细胞的分离被绘制血样后立即进行和嗜中性粒细胞分离后立即成像。 总之，针对烟曲霉中描述了一种下一代活细胞成像技术来非常详细吞噬细胞的抗真菌活性评估。这项技术可以在对黑早期先天免疫防御提供了一个独特的见解单个细胞真菌的相互作用。

烟曲霉是一种条件真菌病原体，并在免疫受损宿主1,2侵袭性肺感染的最常见的真菌病因。理解宿主免疫细胞如何识别和消除黑是真菌研究的一个重要领域，然而，当前可用的杀菌试验有显著的局限性。测量的杀真菌活性的常用的方法包括比色测定和菌落形成单位3,4判定。然而，这些方法在一个单一的时间点，而不是观看主机和病原体之间的动态相互作用提供对真菌存活的信息。因此，真菌是否已被杀害或简单（临时）限制在生长或代谢活性，这些方法都没有考虑到。我们已经开发出能够观测fungici的方法直接DAL活性，同时同时捕获关于吞噬作用，吞噬细胞迁移和真菌生长的抑制的详细信息。 先前，协议，使用实时成像如由小鼠巨噬细胞系5以测量白色念珠菌的吞噬作用的装置发布。这个概念现在已经开发了进一步利用荧光记者曲霉 （FLARE）分生孢子6，7，8，以解决我们的免疫细胞曲霉相互作用的了解的知识差距。这些分生孢子表达红色荧光团（dsRed）组成的和标记有第二荧光团（Alexa氟633）。一旦FLARE分生孢子被杀害，它将停止生产DSRED，其余DSRED变淡，而AF633保持荧光并将其绑定到分生孢子。这将创建与活的和死夫有着明显的区别期间活细胞成像的细胞的NGAL和流式细胞术，并能够以下它们与免疫细胞相互作用个体分生孢子的命运的跟踪。 所描述的测定法提供可视化宿主免疫细胞和曲霉属之间的相互作用的有效方法，并且将在解开负责天然免疫细胞的抗真菌效应物机制的细胞信号传导途径是相当大的价值。其它应用包括可视化变化曲霉生长，如从静息到肿分生孢子，或从肿分生孢子到菌丝开关，如何影响吞噬细胞识别和功能。 以下方案详细描述了如何获得人单核细胞和嗜中性粒如何准备FLARE分生孢子;如何捕捉他们的回答与视频显微镜和流式细胞仪。虽然使用捐助血液中分离人体免疫细胞的在这里，这说明技术已使用各种鼠细胞群的证明同样有效。

<table border="0" cellpadding="0" cellspacing="0" width="913">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">G6279</td>
			<td style="width:411px;">http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T75 flask</td>
			<td style="width:167px;">Greiner Bio-One</td>
			<td>658 175</td>
			<td>http://www.greinerbioone.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:167px;">Gibco</td>
			<td>20012-019</td>
			<td>https://www.thermofisher.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween-80</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td>10592955</td>
			<td>https://nl.fishersci.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40 &#181;m filter</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22363547</td>
			<td>https://www.thermofisher.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml Falcon tube</td>
			<td style="width:167px;">Greiner Bio-One</td>
			<td>188 261</td>
			<td>http://www.greinerbioone.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml Falcon tube</td>
			<td style="width:167px;">Greiner Bio-One</td>
			<td>227 261</td>
			<td>http://www.greinerbioone.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MilliQ</td>
			<td style="width:167px;">Millipore</td>
			<td style="width:119px;">QGARD00R1</td>
			<td>Nanopure water works similarly. http://www.merckmillipore.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biotin</td>
			<td>Molecular Probes</td>
			<td style="width:119px;">B-6352</td>
			<td>https://www.thermofisher.com/uk/en/home/brands/molecular-probes.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">D5879</td>
			<td style="width:411px;">http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptavidin-AF633</td>
			<td>Molecular Probes&#160;</td>
			<td style="width:119px;">S-21375</td>
			<td>AF633 is the only tested fluorophore so far that remains visible in phagosomes. https://www.thermofisher.com/uk/en/home/brands/molecular-probes.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA blood tubes</td>
			<td style="width:167px;">Greiner Bio-One</td>
			<td>455036</td>
			<td>Any blood tubes with an anti-coagulating agent should work. http://www.greinerbioone.com/en/start/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td style="width:119px;">17-1440-03</td>
			<td>http://www3.gehealthcare.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SV3008401</td>
			<td>https://www.thermofisher.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HBSS</td>
			<td style="width:167px;">Gibco</td>
			<td>14170112&#160;</td>
			<td>https://www.thermofisher.com/uk/en/home/brands/gibco.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:216px;">CD14 microbeads</td>
			<td style="width:167px;">Myltenyi Biotec</td>
			<td style="width:119px;">130-050-201</td>
			<td>Other monocyte isolation methods can be used as well, we prefer this method as it gives a consistent high purity of monocytes without being labor intensive. http://www.miltenyibiotec.com/en/</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:216px;">MS Columns</td>
			<td style="width:167px;">Myltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td>See comment at CD14 microbeads. http://www.miltenyibiotec.com/en/</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RPMI + Glutamax</td>
			<td style="width:167px;">Gibco</td>
			<td>72400-021</td>
			<td style="width:411px;">https://www.thermofisher.com/uk/en/home/brands/gibco.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CO2 independent medium</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td>18045054</td>
			<td>Necessary if there is not sufficient CO2 exchange during imaging. https://www.thermofisher.com/uk/en/home</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#181;-slide 8 well glass bottom</td>
			<td style="width:167px;">Ibidi</td>
			<td style="width:119px;">80827</td>
			<td>This is the imaging dish that we use, however any glass imaging dish of proper size should work. http://ibidi.com/</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UltraVIEW VoX 3D Live Cell Imaging System</td>
			<td style="width:167px;">Perkin Elmer</td>
			<td style="width:119px;">L7267000</td>
			<td>Includes volocity software for acquisition and analysis. http://www.perkinelmer.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcofluor white</td>
			<td style="width:167px;">Sigma</td>
			<td>18909</td>
			<td style="width:411px;">http://www.sigmaaldrich.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Voriconazole</td>
			<td style="width:167px;">Sigma</td>
			<td>PZ0005</td>
			<td style="width:411px;">http://www.sigmaaldrich.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD LSRII</td>
			<td style="width:167px;">BD Biosciences</td>
			<td></td>
			<td>Flowcytometer used. https://www.bdbiosciences.com/</td>
		</tr>
	</tbody>
</table>

live imaging of antifungal activity by human primary neutrophils and monocytes in response to a. fumigatus

烟曲霉是一种条件真菌病原体引起免疫妥协的宿主侵入性感染具有高的病死率。研究调查对烟曲霉免疫反应已经缺乏体外测定特异性免疫细胞的抗真菌活性一致和可靠的试验的限制。描述了一种新的方法来评估主单核细胞和从对烟曲霉的人供体使用荧光曲霉记者（FLARE）分生孢子嗜中性粒细胞的抗真菌活性。这些分生孢子含有遗传编码的DsRed记者，其组成由活FLARE分生孢子表达，并用Alexa氟633，这是吞噬溶酶体内降解具有抗性，从而使之间存活和死亡的烟曲霉分生孢子区别是外部标记。视频显微镜，流式细胞仪随后被用于可视化互动分生孢子和先天免疫细胞之间的离子，评估杀真菌活性，同时还对吞噬细胞迁移，吞噬作用和真菌生长的抑制提供了丰富的信息。这种新颖的技术已经提供了令人兴奋的新见解对烟曲霉的主要免疫细胞的宿主-病原体相互作用。要注意执行该法，包括必要的显微镜和流式细胞仪设备所需的实验室设置，并与人供体的血液和基因操纵真菌工作能力是很重要的。然而，该测定能够产生大量的数据，并能揭示详细见解抗真菌响应。该协议已经被成功地用于研究针对烟曲霉初级免疫细胞的宿主-病原体的相互作用。 要注意进行这一测定所需的实验室设置，包括必要的显微镜和流式细胞仪F IT是非常重要的教学设施，并有能力与人类供体血液和遗传操作的真菌工作。然而，该测定能够产生大量的数据，并能揭示详细见解抗真菌响应。该协议已经被成功地用于研究针对烟曲霉初级免疫细胞的宿主-病原体的相互作用。

代表性的结果示下面所描述的协议。 图1示出与人类嗜中性粒细胞和FLARE分生孢子的代表性6小时的活细胞视频。红色荧光蛋白（红色）是由分生孢子自己，而他们已经用AF633（品红）表示。 图2是从从类似电影的单个时间点的图像作为显示在图1中 ，示出了间活的和死的FLARE分生孢子的差异。死分生孢子由失去他们的红色荧光蛋白（红色）信号，同时保持明亮AF633（品红色）的荧光从活分生孢子区分。 烟曲霉分生孢子经常吞噬超过6小时内生存。因此，为了有效杀死量化，流式细胞术示出了用于在图3中16小时温育期图。用一个肺泡巨噬细胞CE获得该数据LL线作为一个例子，但可以使用任何细胞类型中进行。 图4中的迁移显示在刺激人中性粒细胞，以及它们的速度和方向运动的第一个小时。 图5示出已摄取1个，2个，3个或更多个分生孢子而图6显示了萌发成菌丝分生孢子的数目嗜中性粒细胞和单核细胞的百分比。 <img alt="图1" src="/files/ftp_upload/55444/55444fig1.jpg" /> 图1:人嗜中性粒细胞和FLARE分生孢子的活细胞视频显微镜电影。嗜中性粒细胞从人血液中分离，并与在CO 2独立介质FLARE分生孢子以1:1的比率接种一起:3。成像是在37℃下直接发起了与一旋转盘共聚焦显微镜。图像是在1分钟和55秒的间隔在一段6小时来捕获。比例尺=10μm以下。甲在视频放大示出了具有分离的，以澄清在左上角与DIC的FLARE分生孢子的作用通道，红色荧光蛋白（红色）在右上角，AF633（绿色）在左下方和右下方的合并的视频。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/Fig_1.avi">请点击这里下载该视频。</a> <img alt="图2" src="/files/ftp_upload/55444/55444fig2.jpg" /> 图2:单一时间点图像展示之间活的和死FLARE分生孢子的差异。人中性粒细胞用FLARE分生孢子刺激，并用旋转盘共聚焦显微镜成像。图像从生成的电影拍摄。同时保持其AF633荧光（绿色:左下）信号:死孢子从菌丝由已经失去了他们的红色荧光蛋白（右上红）区别开来。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/55444fig2large.jpg">请点击此处</a><html>查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55444/55444fig3.jpg" /> 图3:量化与吞噬和烟曲霉分生孢子杀害验证。鼠肺泡巨噬细胞（MH-S）在一个1 FLARE分生孢子孵育:1的比例在伏立康唑的存在下16个小时。与活分生孢子相关MH-S细胞中示出了红色栅极（DsRed的+ AF633 +）。与死分生孢子相关MH-S细胞中示出了蓝色栅极（DsRed的-AF633 +）。旁观者MH-S细胞中示出了金栅极。卡尔科弗卢尔白，结合真菌细胞壁的细胞不可渗透的荧光染料，用于区分细胞外分生孢子从细胞内的分生孢子（卡尔科弗卢尔白+）（卡尔科弗卢尔白色 - ）。作为一个额外的验证的方法，它被示为具有2μM细胞松弛素D中治疗抑制分生孢子摄取MH-S细胞。e.jove.com/files/ftp_upload/55444/55444fig3large.jpg&quot;&gt;Please点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55444/55444fig4.jpg" /> 图4:人嗜中性白细胞和抗烟曲霉的分生孢子的单核细胞迁移。嗜中性粒细胞从人血液中分离，并与在CO 2独立介质FLARE分生孢子以1:1的比率接种一起:3。成像是在37℃下直接发起了与一旋转盘共聚焦显微镜。图像是在1分钟和55秒的间隔在6小时的时间捕获。每场的所有单独的人中性粒细胞迁移的手动跟踪的使用跟踪软件对静息（A）和肿胀（B）分生孢子1个小时。数据表示为相同的供体的各条件1帧单元。曲折因子（C），的directio的量度纳尔运动和速度（D）然后，使用相同的软件进行定量。平均值±SEM对被示出具有每个供体30个细胞在2个独立实验3个供体。 *:P &lt;0.05（韦尔奇校正t检验）。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/55444fig4large.jpg">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55444/55444fig5.jpg" /> 图5:由人嗜中性白细胞和单核细胞，烟曲霉的分生孢子的吞噬作用。分别为3，:嗜中性粒细胞和单核细胞是从人血液中分离，并与在CO 2独立介质FLARE分生孢子接种在一起的比例为1。成像是在37℃下直接发起了与一旋转盘共聚焦显微镜。图像是在1分钟和55秒的间隔在一段6小时来捕获。吞噬作用测定为细胞含有的量刺激4个小时后荷兰国际集团FLARE分生孢子。数据被呈现为平均值±SEM从3个供体和在2个独立实验每个供体3帧。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/55444fig5large.jpg">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55444/55444fig6.jpg" /> 图6: 由嗜中性粒细胞和单核细胞 ，烟曲霉 分生孢子发芽的抑制作用 。分别为3，:嗜中性粒细胞和单核细胞是从人血液中分离，并与在CO 2独立介质FLARE分生孢子接种在一起的比例为1。成像是在37℃下直接发起了与一旋转盘共聚焦显微镜。图像是在1分钟和55秒的间隔在6小时的时间捕获。真菌萌发的抑制是通过测量分生孢子是有细菌的百分比检查后共培养2,4和6小时inated。数据被呈现为平均值±SEM从3个供体和在2个独立实验每个供体3帧。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/55444fig6large.jpg">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus at JoVE.com

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

在这里，我们描述了一种协议与活细胞视频显微结合使用荧光曲霉分生孢子记者评估初级人类免疫细胞的抗真菌活性在实时和流式细胞术。所生成的数据提供深入了解宿主细胞曲霉相互作用如杀真菌活性，吞噬作用，细胞迁移和真菌生长的抑制。

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

live-imaging-antifungal-activity-human-primary-neutrophils-monocytes

Research

JoVE Journal

Immunology and Infection

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

10.1K 次观看.  University of Aberdeen. 在这里，我们描述了一种协议与活细胞视频显微结合使用荧光曲霉分生孢子记者评估初级人类免疫细胞的抗真菌活性在实时和流式细胞术。所生成的数据提供深入了解宿主细胞曲霉相互作用如杀真菌活性，吞噬作用，细胞迁移和真菌生长的抑制。 

视频: Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...