这项研究是由美国国立卫生研究院R00HL102154支持，以及血液学学者奖的美国社会为P吉。

在此协议中描述的实验是在按照规定由美国西北大学实验动物管理和使用委员会的指导方针和规定执行。 1，制备培养基<ol><li>准备纤维连接蛋白的解决方案。加入1毫升水，以人纤连蛋白（1毫克）中的一个小瓶内。离开溶液在组织培养橱中30分钟而不搅拌。总的液体转移至50ml的PBS中，以20微克/毫升的最终浓度，并轻轻混匀。使涂覆的板（见下文）之前的纤连蛋白的溶液新鲜。 </li><li>准备50毫升促红细胞生成素的培养基（SCF介质）。结合所列出的成分在表1中的介质可以被存储在4℃下1个月时发劲。 </li><li>准备50毫升含有促红细胞生成素的培养基。结合所列出的成分在表2中的介质可以被存储在4℃下1个月时发劲。 </li></OL&gt; 2，纤维连接蛋白涂层板<ol><li>加入1 ml纤连蛋白溶液到6孔板（或0.5毫升至12孔板的每孔）的每个孔中。离开该板在组织培养罩1小时。 </li><li>冲洗孔中，用无菌水洗涤两次，并空气干燥该板。 </li><li>包装板，并保存在寒冷的房间在4℃。 </li></ol> 3，净化胎肝红细胞的注意：使用从E13到E15胎肝定时妊娠小鼠（C57BL / 6）。 E13.5肝脏是首选，以最大限度地提高CFU-E祖细胞的产率。以获得定时怀孕小鼠，6〜8周龄的雄性和雌性小鼠放置在一个笼（通常是一个男性与一个或两个雌性）。第二天早晨女性阴道塞被检查，以确定是否交配是否成功。怀孕的第一天是当天被发现后插上。 <ol><li>总胎肝细胞的制备<ol><li>牺牲谅解备忘录SE的CO 2吸入和颈椎脱位。喷用70％乙醇消毒腹部。用解剖剪刀切除子宫打开腹部。洗1X PBS子宫。 </li><li>解剖用组织培养罩和高压手段在无菌条件下将胎儿和洗1X PBS中。 </li><li>从解剖胎儿胎儿的肝脏。握住胎儿的身体有一个镊子，轻轻一拉胎肝（粉红色）远离身体与另一个钳。用钳子清洁胎儿肝脏以除去相关的纤维化组织。将所有的胎儿肝脏到新鲜的1×PBS含有10％胎牛血清。 </li><li>机械地上下吹打破坏胎肝。 </li><li>过滤通过40微米的细胞过滤的细胞悬浮液置于50ml锥形管中。沉淀细胞，在800×g离心5分钟。 </li><li>吸出上清液并将细胞重新悬浮于1ml PBS中，用10％的FBS（约8 E13.5胎肝每毫升）。 </li></ol></li><li>的TER119负红细胞的净化<ol><li>方框在冰上将细胞用大鼠IgG（0.2毫克/毫升）15分钟。 </li><li>未经洗涤或离心分离，生物素化的抗TER119抗体加入到细胞悬浮液（1微克/毫升）。孵育在冰上15分钟。 </li><li>在1×PBS中，用10％的FBS（1:10）和离心机在800×g离心5分钟，洗净。重悬在1ml的PBS中，用10％FBS的细胞。 </li><li>加入75微升链霉结合有磁性粒子和孵育10分钟在冰上。 </li><li>的体积加入到2.5毫升的PBS含10％FBS。细胞悬液转移到5ml聚丙烯圆底试管中。 </li><li>插入管插入的磁性细胞分选装置，并孵育10分钟。 TER119阳性细胞将附着在管的一侧。未附着的TER119阴性红细胞将保持在悬浮液中。 </li><li>倒入细胞悬液到一个新的5毫升聚丙烯圆底试管。 </li><li><html>重复步骤3.2.6和3.2.7，除去残留的TER119阳性细胞。 </li><li>沉淀细胞，在800×g离心5分钟。抽吸掉上清液。重悬细胞于1ml PBS中含有10％FBS和计数活细胞用台盼蓝。 </li></ol></li></ol> 4，转导与病毒编码基因或shRNA <ol><li>板式纯化TER119负胎儿肝细胞在3-5×10 5个/孔在包被12孔板纤连蛋白（如果使用一个不同的板调整细胞数）。 </li><li>加入聚凝胺至10微克/毫升的最终浓度，以促进病毒转导。自旋感染细胞用慢病毒或逆转录病毒，编码的cDNA或shRNA，以800×g离心1-1.5小时，在37℃。 </li></ol>转导TER119负鼠胎肝红细胞的5文化<ol><li>来测试基因功能的终端红细胞生成的早期阶段（ 图1中虚线）。“&gt; </ol.><ol><li><ol. li="">培养的转导的细胞中促红细胞生成素的培养基（SCF介质）12小时，以允许的转基因和维护祖状态的表达。 </ol.></li><li>离心细胞，在800×g离心5分钟。吸出上清液。悬浮介质收容在一个12孔板的每个孔中的细胞在1ml生成素。 </li><li>培养24或48小时后，收获细胞用于进一步分析。 </li></ol></li><li>测试基因功能的终端的红细胞生成（ 图1中的实线）的后期阶段。 <ol><li>文化的转立刻细胞生成素含有中。 </li><li>培养24或48小时后，收获细胞用于进一步的分析。 </li></ol></li></ol> 6染色的培养胎肝细胞的流式细胞仪分析<ol><li>清洗和悬浮细胞在1X PBS。收获的2×10 5细胞培养于流式细胞仪分析。 </li><li>孵育将细胞与荧光标记的抗体20分钟，在室温下在黑暗中。为了测试分化，使用的APC标记的抗CD71和PE标记的抗TER119。为了测试去核，用Ho​​echst的33342染色，除了其他的抗体。 </li><li>用1×PBS洗涤细胞。重悬含有1％FBS和碘化丙啶（PI）（1微克/毫升）进行染色的死细胞，并从分析中排除它们的细胞在0.5ml PBS中。 </li><li>开展流动cytrometric分析。首先调整和补偿流式细胞仪的分析单色染色和未染色的对照细胞。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

在这里，我们提出了一个独特的系统，按时间顺序分析小鼠胎肝终端红细胞生成。通过对不同培养条件下的应用，我们成功地解剖在早期和晚期终端红细胞生成。这是特别重要的，以确定基因与多种功能的机制。例如，外消旋GTP酶发挥终端红细胞生成的不同阶段起重要作用。 Rac的GTP酶在终端红细胞生成影响细胞分化和增殖的早期阶段抑制。在另一方面，抑制Rac的GTP酶的活性，在终端红细胞生成块...

红血球生成是多能造血干细胞的成熟红细胞的分化过程。此分步处理包括类红细胞爆形成单位（BFU-ES），在快速分裂的红细胞集落形成单位（CFU-ES），及形态识别红细胞1,2的形成。由CFU-E祖细胞终端红细胞生成包括连续的促红细胞生成素依赖性和独立的阶段2,3。在终端红细胞生成的早期阶段，促红细胞生成素（EPO）结合到其细胞表面上的受体，并诱导一系列下游信号通路，防止细胞凋亡，促进细胞快速分裂和基因表达1,4。在终端红细胞生成的后期，有核红细胞进行终端细胞周期出口，染色质和核缩合，并挤出的高度浓缩的细胞核分 。 我们的终端的理解红细胞生成极大，在过去的几十年中，这主要是由于在体外成功地利用一些和体内小鼠模型6-9改善。在这些模型中， 在小鼠胚胎肝红细胞体外培养提供了许多优点，包括易于细胞纯化，快速增殖和分化，并更密切模仿人类红细胞生成10,11。在这个系统中，大量的从鼠胎肝红系祖细胞可以容易地由TER119的单步骤纯化（标记为成熟红细胞）阴性红细胞分离。中的红细胞的两天的培养，这些细胞的分化可以通过基于所述转铁蛋白受体（CD71）和TER119抗原12的表面表达的流式细胞术分析来监测。此外，晚期红细胞分化的眼球摘除可以用DNA制造商（赫斯特33342）被检测到13，此外，纯化的祖细胞可被转用的cDNA或小发夹RNA（shRNA的）感兴趣的基因，这有利于基因表达的功能的机理研究对红血球生成11,13,14的外源表达修饰。 在另一方面，快速的细胞生长速度可以是一把双刃剑，因为它很难在终端红细胞生成的不同阶段来描述基因功能。在大多数情况下，很难确定是否在终端红细胞生成的早期阶段的特定基因的功能，因为通过时间的cDNA或shRNA的表达，所述细胞已经通过了早期阶段。为了解决这个问题，我们开发了一个独特的系统剖析终端红细胞生成的早期和晚期阶段。对于终端红细胞生成的早期阶段，转基因TER119阴性红细胞培养在促红细胞生成素的培养基中，但含有干细胞因子（SCF），白细胞介素-6和FLT3配体以维持其祖的地位，并允许转导的cDNA和shRNA的表达13。将细胞改变到促红细胞生成素含培养基12小时后，诱导细胞增殖和分化。在这种方式中，当细胞开始分化，在转导的cDNA或shRNA的已经表达出来。对于终端红细胞生成后期，TER119负红细胞人转导后，立即含中Epo的培养。因此，可以分析在终端红细胞生成的后期所感兴趣的基因的功能。综上所述，本系统的广泛应用，将有助于终端红细胞生成的不同阶段解剖基因的功能。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s MediumIMDM (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GEMINI Bio-product</td>
			<td>700-102P</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mecaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>0.1 M in IMDM,&#160; 4&#8451;stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Hyclone</td>
			<td>SH30034.01</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Stem cell factor (SCF)</td>
			<td>STEMCELL TECH</td>
			<td>2630</td>
			<td>100 ng/ul, -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Mouse FIT3 Ligand (Flt-3L)</td>
			<td>BD Biosciences&#160;</td>
			<td>14-8001-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Recombinant Interleukin-6 (IL-6)&#160;</td>
			<td>GEMINI Bio-product</td>
			<td>300-327P</td>
			<td></td>
		</tr>
		<tr>
			<td>fibronectin</td>
			<td>BD Biosciences&#160;</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>STEMCELL TECH</td>
			<td>9300</td>
			<td>10% stock in IMDM</td>
		</tr>
		<tr>
			<td>Insulin solution, human</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>4&#8451; stock</td>
		</tr>
		<tr>
			<td>holo-transferrin human</td>
			<td>Sigma</td>
			<td>T0665</td>
			<td>50 mg/ml in Water -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Erythropoietin(Epo)</td>
			<td>PROCRIT</td>
			<td>NOC 59676-303-00</td>
			<td>3,000 U/ml stock</td>
		</tr>
		<tr>
			<td>Streptavidin particles Plus</td>
			<td>BD Pharmingen</td>
			<td>557812</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>STEMCELL TECH</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round-Bottom Tube, 5 ml</td>
			<td>FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

包括红细胞生成一个动态的过程，开始于随后迅速分裂红系集落形成单位（CFU-ES）致力于红爆形成单位（BFU-ES）。后CFU-ES，细胞形态识别，一般称为终端红细胞。其中一个终端红细胞的研究挑战是缺乏实验方法来剖析基因功能以时间的方式。在这个协议中，我们描述了一种独特的策略，以确定在终端红细胞生成的早期和晚期基因功能。在这个系统中，小鼠胎肝TER119（成熟红细胞细胞标记物）阴性红细胞中纯化和转导的cDNA或小发夹RNA（shRNA的）感兴趣的基因的外源表达。随后将细胞培养在含有培养基比红细胞生成素（EPO）等，以保持它们的祖细胞阶段为12小时，同时使外源的cDNA或shRNA的生长因子来表达。将细胞改变到促红细胞生成素培养基中12小时后，诱导细胞分化和增殖，而已经表达了外源遗传物质。这个协议有助于在终端红细胞生成的早期阶段的基因的功能分析。研究后期终端红细胞生成，细胞培养，立即在转导后生成素中。在这种方式中，细胞已分化到终端红细胞生成的后期时进行表达的转基因材料。我们建议使用此策略的普遍应用，这将有助于了解在终端红细胞生成的不同阶段，详细的基因功能。

图1列出了实验策略。该协议包括两个独立的条件为靶向的信号分子的功能的终端红细胞生成的早期和晚期阶段。 TER119负胎肝红细胞从E13.5小鼠胚胎纯化。胎肝类红细胞前和纯化后的流式细胞术分析表明，纯化效率高（ 图2A和2B）。对于终端红细胞生成（ 图1中，虚线）的早期阶段，病毒转导后，将细胞在红细胞生成素的培养基（SCF介质）12小时进...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

我们提出了一个在体外小鼠胎肝红细胞培养系统剖析终端红细胞生成的早期和晚期阶段。这个系统有利于在不同的发育阶段特异性基因的功能分析。

小鼠胎肝文化系统解剖靶基因功能的终端红细胞生成的早期和晚期

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

SCIENTISTS IN ACTION

14.8K 次观看.  Northwestern University. 我们提出了一个在体外小鼠胎肝红细胞培养系统剖析终端红细胞生成的早期和晚期阶段。这个系统有利于在不同的发育阶段特异性基因的功能分析。 

视频: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

牛痘病毒记者量化病毒功能在基因表达的各个阶段

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...