这项工作得到了创新的年轻生物技术专家奖BT/BI/12/040/2005和BT/PR13289/BRB/10/745/2009赠款生物技术，印度和中心的DNA指纹图谱和诊断核心资金的政府部门的支持，海得拉巴。 MNR和GB是初中和科学与工业研究理事会对追求马尼帕尔大学博士学位的高级研究奖学金的受助人。 SB是初中和生物技术部高级研究奖学金朝着追求马尼帕尔大学的博士学位获得者。

建议在执行C。光滑念珠菌感染的实验与生物安全防护水平的2实验室（BSL-2）。 <ol><li>制备THP-1巨噬细胞单层。 </li><li> C.编制光滑念珠菌细胞悬液。 </li><li> THP-1巨噬细胞C的感染光滑的细胞。 </li><li>测量通过集落形成单位测定吞噬率和细胞内复制。 </li><li>监控用共聚焦激光扫描显微镜细胞内复制。 </li></ol> 1。 THP-1巨噬细胞单分子膜的制备<ol><li> THP-1是一个1岁男孩患急性单核细胞白血病14痛苦的外周血来源的人类单核细胞系。 THP-1中，悬浮细胞系，是经常在潮湿的5％CO 2中在补充有10％FBS（胎牛血清）的RPMI-1640培养基中维持在37℃。治疗与佛波醇12 - myristaTE 13 - 乙酸酯（PMA，佛波酯）导致的THP-1单核细胞的终末分化成巨噬细胞。重要的是，PMA处理不影响细胞活力及分化的细胞不分裂。 </li><li>种子约1.5×10 6 THP-1单核细胞在100毫米的培养皿中，在RPMI-1640完全培养基（RPMI-1640培养基补充有10％血清，2mM谷氨酰胺和1x抗生素;为10ml /皿），并让细胞生长在37 °℃，5％CO 2的2天。 </li><li>一旦THP-1细胞已经达到了8×10 5细胞/ ml的密度，将培养皿转移至细胞培养物层流罩，轻轻地摇动它重新悬浮沉淀的细胞。 </li><li>吸取细胞悬液出来，放入15毫升无菌试管中并离心分离机以1000rpm（130×g离心）4分钟。弃上清，并暂停THP-1细胞沉淀在5ml新鲜，预热的培养基，RPMI-1640完全培养基中。 </li><li>列举细胞用血细胞计数器和稀的大公升悬浮至10 6个细胞/ ml用预热的RPMI-1640完全培养基中的终浓度</li><li>加入1μl的160毫米PMA股票（佛波醇12 - 十四烷酸酯-13 - 乙酸酯溶液制备的二甲基亚砜）至10 ml THP-1细胞悬液（终浓度为16纳米）拌匀。分配1毫升细胞悬液到24孔组织培养板的各孔中，并在保持在37℃，5％CO 2的12小时培养箱孵育板。 </li><li>除去旧培养基，加入1 ml预热的RPMI-1640完全培养基中，并让细胞恢复12小时，于37℃在5％CO 2。 </li><li>观察在倒置显微镜下观察细胞，以确保椭圆形的THP-1单核细胞的悬浮液已分化为扁平，梭形和附着的巨噬细胞。这些分化的细胞现在已经准备好进行C。光滑念珠菌感染的研究。 </li></ol> 2。 C.编制光滑 ＆＃160;细胞悬浮 光滑念珠菌野生型菌株BG2将被用于感染的THP-1巨噬细胞。C.光滑念珠菌细胞常规培养在液体YPD（酵母提取物（1％）-蛋白胨（2％）-葡萄糖（2％））培养基中。固体YPD培养基中，加入2％的琼脂培养基在高压灭菌前制备。 <ol><li>为了准备C.文化光滑念珠菌 ，挑取单个菌落从琼脂平板上，用无菌的，一次性环，接种到10ml的YPD液体培养基和14-16小时，孵育在30℃下振荡（200rpm）下</li><li>离心该培养（1毫升）在4,000 rpm（1,800×g离心）5分钟，在台式微量离心机，三次洗涤细胞，用无菌PBS（磷酸盐缓冲盐水，pH 7.4）中，并暂停细胞沉淀在1ml PBS中。 </li><li> C.测量外径600 光滑念珠菌细胞悬浮液，并用无菌PBS中适当体积稀释到获得的2×10 6个细胞/ ml的密度（OD 600= 0.1）。或者，所需的细胞密度可以通过使用血球计数酵母细胞来实现。 </li></ol> 3。的THP-1巨噬细胞与C感染光滑念珠菌细胞<ol><li>加入50微升C。光滑念珠菌细胞悬浮液以THP-1巨噬细胞（每24孔培养板的井10 6个细胞接种），得到的MOI为0.1（感染复数）孵育板在37℃下用5％的CO 2。 </li><li>为了确定可行的酵母计数，稀释50微升C.光滑念珠菌细胞悬液100倍的无菌PBS和板100微升在YPD固体培养基。孵育板在30°C和手工清点后1-2天出现酵母菌菌落数。这些数字将，从此，被称为0小时C。光滑的CFUs（菌落形成单位）。 </li><li>后的THP-1细胞与C的2小时共培养通过反相24孔培养板光滑细胞，弃培养基我呐水库和轻轻地洗三次，用预热无菌PBS去除nonphagocytosed外三光滑的细胞。轻柔洗涤，用PBS是绝对必要的，以实现完全除去所有细胞外的酵母而不会造成对THP-1巨噬细胞单层的任何损坏。 </li></ol> 4。通过集落形成单位测定吞噬率和细胞内复制的测量<ol><li>裂解C。光滑念珠菌感染的THP-1巨噬细胞以1毫升无菌H 2 O的2分钟，轻轻刮掉，除去从井的巨噬细胞碎片和在微量离心管收集细胞裂解液。的完整的巨噬细胞裂解微观验证是举足轻重的所有内在化酵母的回收​​。 </li><li>制备100倍稀释的溶胞产物的无菌PBS，板100微升在YPD固体培养基中孵育板在30℃下</li><li> 1-2天后，手动计数C的数光滑念珠菌菌落APpeared YPD培养基上，乘以稀释倍数（2小时的CFU），并计算吞噬率是指C的百分比是由THP-1巨噬细胞后2小时共同孵育使用下列公式计算摄入的光滑念珠菌细胞。 ％吞噬= [（CFU的在2小时）/（CFU的，在0小时）]×100 </li><li>测量C的胞内复制的速率光滑念珠菌细胞在THP-1巨噬细胞，在不同时间点感染后，即收集细胞酵母。 4，6，8，10，12，和24小时，通过重复步骤3.3-4.3。 </li><li>计算C的折叠生存/复制光滑念珠菌细胞在THP-1巨噬细胞中通过将总的CFUs在任何给定时间点与那些在2小时（吞噬的酵母）。 </li></ol> 5。细胞内复制的使用共焦拉斯监控呃扫描显微镜<ol><li>以下在第1节中所述的步骤，种子PMA处理的5×10 5个THP-1细胞中的2 4 -孔室玻片各孔中，在37℃，5％CO 2下12小时。 </li><li>更换旧培养基用预热的RPMI-1640完全培养基中和允许细胞从PMA处理恢复12小时。 </li><li>制备GFP（绿色荧光蛋白）标记的C.光滑念珠菌细胞悬浮液作为第2节所述，感染到的THP-1巨噬细胞为1的MOI。如果C。光滑念珠菌菌株表达GFP不可用，用FITC标记的（荧光素异硫氰酸酯）的酵母细胞，可用于监测早期事件感染后即 。吞噬率和phagolysosomal成熟时2小时。 </li><li>孵育玻片2小时，在37℃，5％CO 2，反转它们仔细地丢弃培养基，用无菌洗3次，预热的PBS中。 </li><li>加入500μl预热的RPMI-1640完全的Medi微米至1滑动的各腔室，并用5％CO 2的22小时孵育它在37℃。 </li><li>要解决2小时C。光滑念珠菌感染的THP-1巨噬细胞，加入500μl的3.7％甲醛（在PBS中制备的）的其他幻灯片的各腔室和孵化它在室温下放置20分钟，在黑暗中。 </li><li>洗净用PBS三次滑动，加入500μl的Triton-X（0.7％），并在室温下孵育5分钟，在黑暗中。 </li><li>滑洗3次，用PBS，从文化的幻灯片中删除室和空气干燥在黑暗中3-5分钟。 </li><li>使用含DAPI的Vectashield封固剂（4&#39;，6 - 二脒基-2 - 苯基吲哚）上滑动，避免形成气泡小心装入盖玻片。轻轻地取出多余的液体用的Kimwipe和密封盖玻片的边缘用指甲油。在黑暗中，在4°C，直到使用存储的幻灯片。 </li><li>重复步骤5.4和5.6-5.9处理THP-1细胞24小时后的感染。 </li><li>使用激光扫描confo细胞图像卡尔显微镜（60X油浸物镜，激发波长为405纳米和488纳米的DAPI和GFP染色，分别）。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

先天免疫系统中起着的机会性真菌感染的控制中起重要作用。巨噬细胞促进食入​​和破坏病原真菌的抗真菌防御。因此，所需要的生存和/或对抗巨噬细胞的抗菌功能的因素，阐明将增进我们对真菌毒力的战略认识。在这方面，我们已经建立了使用来自人单核细胞系THP-1巨噬细胞以表征一个人的机会性真菌病原体C的相互作用在体外细胞培养模型系统光滑念珠菌与宿主吞噬细胞...

念珠菌是免疫功能低下患者1危及生命的侵袭性真菌感染的主要原因。 光滑念珠菌 ，一个新兴的院内病原体，是从重症监护病房的患者取决于地理位置1-3第二个或第三个最常见的孤立念珠菌。系统发育，C.光滑念珠菌 ，单倍体芽殖酵母，更密切相关的非致病模型酿酒酵母不是致病念珠菌。包括C。白色念珠菌 4。与此相一致，C。光滑缺少一些关键的真菌毒力性状，包括交配，分泌的蛋白水解活性和形态可塑性4-5。 虽然C。光滑不形成菌丝，它可以生存和复制在小鼠和人类巨噬细胞6-8这表明它已经开发了独特的发病机制。有限的信息，请访问有关的策略，C.采用光滑的生存贫营养的巨噬细胞内环境，抵制氧化和挂载宿主免疫细胞5非氧化宿主反应。一个与此相关巨噬细胞模型系统是一个先决条件划定C的相互作用光滑念珠菌与宿主吞噬细胞通过功能基因组学和蛋白质组学的方法。外周血单核细胞（PBMC）和人及鼠源的骨髓衍生的巨噬细胞（BMDMs），分别刚才被用来研究C的相互作用光滑念珠菌与宿主免疫细胞7,9。然而，难以获得外周血单个核细胞和BMDMs，他们有限的生命跨度和不同哺乳动物捐助者之间的内在差异限制了这些细胞的利用率多才多艺的模型系统。 在这里，我们描述了一种建立在体外系统研究intracellul的C的AR行为光滑念珠菌细胞从人单核细胞系THP-1巨噬细胞。本协议的总体目标是制定可以很容易地操纵研究宿主真菌病原体相互作用的不同方面简单，价格低廉，快速，重现性细胞培养模型系统。 THP-1细胞先前已用于解密对广范围的病原体，包括细菌，病毒，真菌和10-12的宿主免疫应答。单核细胞THP-1细胞易于维护，并且可以区分，在佛波酯治疗，巨噬细胞它模仿人类单核细胞源性巨噬细胞和表达适当的巨噬细胞标记物13。 THP-1巨噬细胞模型系统的主要优点是易于使用性和缺乏先进的设备要求。 这里介绍的协议是很容易适应学习其他人的真菌病原体与居屋的相互作用吨的免疫细胞。当前的过程也可以用来识别毒力因子的感兴趣使用高通量突变体屏幕上的病原体。这证明型概念例证的成功使用THP-1培养模型系统来识别一组在人类巨噬细胞8所需的光滑念珠菌生存的56个基因。

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			<td height="39" style="height:40px;width:163px;">THP-1</td>
			<td style="width:105px;">American Type Culture Collection</td>
			<td style="width:85px;">TIB 202</td>
			<td style="width:243px;">Human acute monocytic leukemia cell line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">RPMI-1640</td>
			<td style="width:105px;">Hyclone</td>
			<td style="width:85px;">SH30096.01</td>
			<td style="width:243px;">For maintaining THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Phorbol 12-myristate 13-acetate</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">P 8139</td>
			<td style="width:243px;">Caution: Hazardous</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">YPD&#160;</td>
			<td style="width:105px;">BD-Difco</td>
			<td style="width:85px;">242710</td>
			<td style="width:243px;">For growing Candida glabrata cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Formaldehyde</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">F8775</td>
			<td style="width:243px;">For fixation of C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Phosphate buffered saline (PBS)</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,&#160; pH 7.4) for washes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Saline-sodium citrate (SSC)&#160;</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (3 M NaCl, 0.3 M sodium citrate for 20x concentration) for washes</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Prehybridization buffer</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (50% formamide, 5x Denhardt&#8217;s solution, 5x SSC, 1% SDS) for hybridization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">VECTASHIELD mounting medium</td>
			<td style="width:105px;">Vector Labs</td>
			<td style="width:85px;">H-1200</td>
			<td style="width:243px;">For mounting slides for confocal microscopy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">32P-labeled &#945;-dCTP</td>
			<td style="width:105px;">JONAKI-BARC</td>
			<td style="width:85px;">LCP-102</td>
			<td style="width:243px;">For radiolabeling of signature tags</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">100 mm tissue culture dishes</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">430167</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">24-well tissue culture plate</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">3527</td>
			<td style="width:243px;">To perform C. glabrata infection studies in THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">4-chamber tissue culture-treated glass slide</td>
			<td style="width:105px;">BD Falcon</td>
			<td style="width:85px;">REF354104</td>
			<td style="width:243px;">To image C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hemocytometer</td>
			<td style="width:105px;">Rohem India</td>
			<td style="width:85px;"></td>
			<td style="width:243px;">For enumeration of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Table top microcentrifuge</td>
			<td style="width:105px;">Beckman Coulter</td>
			<td style="width:85px;">Microfuge 18</td>
			<td style="width:243px;">For spinning down cells in microtubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Table top centrifuge</td>
			<td style="width:105px;">Remi</td>
			<td style="width:85px;">R-8C</td>
			<td style="width:243px;">For spinning down cells in 15 ml tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Spectrophotometer</td>
			<td style="width:105px;">Amersham Biosciences</td>
			<td style="width:85px;">Ultraspec 10</td>
			<td style="width:243px;">To monitor absorbance of yeast cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Plate incubator</td>
			<td style="width:105px;">Labtech</td>
			<td style="width:85px;">Refrigerated</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Shaker incubator</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:85px;">Innova 43</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Water jacketed CO2&#160; incubator</td>
			<td style="width:105px;">Thermo Electron Corporation</td>
			<td style="width:85px;">Forma series 2</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Confocal microscope</td>
			<td style="width:105px;">Carl Ziess</td>
			<td style="width:85px;">Ziess LSM 510 meta&#160;</td>
			<td style="width:243px;">To observe C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Compound microscope</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:85px;">CKX 41</td>
			<td style="width:243px;">To observe C. glabrata and THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">PCR machine</td>
			<td style="width:105px;">BioRad&#160;</td>
			<td style="width:85px;">DNA Engine</td>
			<td style="width:243px;">To amplify unique tags from input and output genomic DNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hybridization oven</td>
			<td style="width:105px;">Labnet</td>
			<td style="width:85px;">Problot 12S</td>
			<td style="width:243px;">For hybridization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">PhosphorImager</td>
			<td style="width:105px;">Fujifilm</td>
			<td style="width:85px;">FLA-9000</td>
			<td style="width:243px;">For scanning hybridized membranes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Thermomixer</td>
			<td style="width:105px;">Eppendorf</td>
			<td style="width:85px;">Thermomixer Comfort</td>
			<td style="width:243px;">For denaturation of radiolabeled signature tags</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:163px;">Gel documentation unit</td>
			<td style="width:105px;">Alphainnontech</td>
			<td style="width:85px;">Alphaimager</td>
			<td style="width:243px;">To visualize ethidium bromide-stained DNA in agarose gels</td>
		</tr>
	</tbody>
</table>

establishment of an in vitro system to study intracellular behavior of candida glabrata in human thp-1 macrophages

一种细胞培养模型系统中，如果主机环境条件密切模仿，可以作为一种廉价的，可再现的和容易可操纵替代动物模型系统中的微生物病原体感染的特定步骤的研究。一个人单核细胞株THP-1其中，经佛波酯治疗，分化为巨噬细胞，以前被用来研究的许多细胞内的病原体包括结核杆菌毒力的战略。在这里，我们讨论的协议制定的体外细胞培养模型使用THP-1巨噬细胞中描绘的人的机会性病原体酵母光滑念珠菌与宿主吞噬细胞的相互作用的系统。该模型系统结构简单，速度快，适合于高通量突变屏幕，并且不需要复杂的设备。一个典型的THP-1巨噬细胞感染实验大约需要24小时一个额外的24-48小时，使细胞内收回酵母生长在营养丰富的培养基进行菌落形成单位为基础的可行性分析。像其他的体外模型系统中，这种方法的一个可能的限制是难以外推得到的一个高度复杂的免疫细胞的电路存在于人类宿主的结果。然而，尽管这样，目前的协议是要阐明病原真菌可以采用回避/抵抗抗生素的反应和生存，适应和增殖的宿主免疫细胞的营养环境恶劣的策略非常有用。

PMA处理的THP-1巨噬细胞C.感染分析光滑念珠菌的野生型（wt）细胞中发现， 重量细胞，巨噬细胞在2小时后共同培养的速率的55-65％，吞噬。此外，C。光滑念珠菌细胞能够由THP-1巨噬细胞的抗杀并进行了适度的5 -至与THP-1巨噬细胞的共培养8的24小时后的7倍增加的CFUs。野生型细胞，转化有表达GFP的质粒，在THP-1巨噬细胞内的复制，也验证了用共聚焦荧光显微镜...

Watch this Scientific Journal Video about Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages at JoVE.com

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

当前文章概述了一个协议来建立体外细胞培养模型系统来研究一种兼人体细胞内的真菌病原体光滑念珠菌与人体巨噬细胞将是一个有用的工具，以增进我们对真菌毒力机制知识的互动。

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

establishment-an-vitro-system-to-study-intracellular-behavior-candida

Research

JoVE Journal

Immunology and Infection

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

11.5K 次观看.  Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India. 当前文章概述了一个协议来建立体外细胞培养模型系统来研究一种兼人体细胞内的真菌病原体光滑念珠菌与人体巨噬细胞将是一个有用的工具，以增进我们对真菌毒力机制知识的互动。 

视频: Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

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 ...

科研

免疫与感染

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...