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通过流式细胞术评价靶标结合<ol><li> 制备生物材料和试剂 <ol><li>制备500mL补充有10％热灭活胎牛血清（H-IFBS）的RPMI培养基。 </li><li>使用RPMI和75-cm 2培养瓶培养Daudi Burkitt&#39;s淋巴瘤（Daudi）细胞和Daudi GFP +细胞。将培养物保持在37℃，5％CO 2加湿气氛中，直到达到6 - 9×10 5个细胞/ mL。 </li><li>通过在PBS中稀释1/100 H-IFBS制备50 mL染色缓冲液;该缓冲液在2-8℃下稳定至少一个月。 </li><li>为参考和生物仿制mAb准备测试解决方案。在染色缓冲液中制备10个1：2系列稀释液（各500μL），从5μg/ mL开始。 </li><li>使用染色缓冲液将人IgG（同种型对照）稀释至5μg/ mL和PE-Cy5小鼠抗人IgG（二抗）至con制造商建议的中心。 </li><li>在PBS（固定缓冲液）中制备4％多聚甲醛。 </li></ol></li><li> 目标绑定 <ol><li>从75 cm 2培养瓶中收集Daudi和Daudi GFP +细胞悬液，并将其转移到15 mL离心管中。以400 xg离心5分钟。 </li><li>通过加入5毫升PBS洗涤细胞，并以400×g离心细胞悬浮液5分钟。 </li><li>将细胞重悬于PBS中，并用台盼蓝进行细胞计数和活力分析。使用细胞活力水平≥95％的培养物进行分析。 </li><li>用冷染色缓冲液将细胞悬浮液稀释至4×10 6个细胞/ mL。 </li><li>在1.5 mL微量离心管中，将50μL细胞悬浮液加入100μL不同测试浓度的参考或生物仿制mAb。包括每个实验条件的重复。 </li><li>准备额外的管同种型对照（人IgG1而不是利妥昔单抗）和阴性对照（无抗体的二抗）。 </li><li>在4℃下孵育20-30分钟。 </li><li>通过加入1mL PBS洗涤细胞，并在10℃下以400×g离心细胞悬浮液5分钟。丢弃上清液。 </li><li>将细胞悬浮在100μL的二抗中，并在4℃下孵育20-30分钟，免受光照。 </li><li>用PBS洗涤细胞两次，并将其悬浮于200μL固定缓冲液中。 </li><li>分析流式细胞仪上的细胞。 注意：如果样品储存在4°C并保护光线，信号保持稳定数天。 </li></ol></li><li> 数据采集 <ol><li>在流式细胞仪操作软件的工作表上打开两个点图。 FSC-A 与 FSC-H在第一和FSC-A与SSA-A中的设置。打开PE-Cy5通道的直方图。 </li><li>在FSC-A与FSC-H图中，制作选择单线事件的门（R1）（ 图1A ）。 </li><li>将FSC-A中的R1群体设置为SSA-A点阵图，然后选择新的门（R2）选择目标单元格（ 图1B ）。设置PE-Cy5强度直方图中的R2群体，以查看细胞的频率分布。 </li><li>使用阴性和同种型对照调节PE-Cy5通道的较低荧光强度（FI）极限（ 图1C ）。 </li><li>从参考产品浓度较高的样品中获取R2内的10,000个事件。该样品的FI应该是预期的最高值（ 图1C ）。 </li><li>获取其余的样品。 </li><li>对于每个样品，得到PE-Cy5通道中的中值荧光强度（MFI）。 </li><li>对于具有参考或生物仿制mAb的样品，计算样品MFI与同种型对照（ΔMFI）的差异。</li></ol></li></ol> 2. CDC的评估<ol><li> 生物材料和试剂的制备 <ol><li>制备细胞培养基和培养的Daudi和Daudi GFP +细胞，如上所述（步骤1.1.1 - 1.1.2）。 注：此外，CDC测定需要无血清RPMI。 </li><li>稀释的正常人血清补体（NHSC）1：2用无血清的RPMI中。准备2.5毫升。 </li><li>制备1毫升的热灭活（30分钟/ 56℃）的NHSC 1:2稀释用RPMI。 </li><li>准备套基准测试解决方案和单克隆抗体生物仿制药在无血清RPMI。使从1 10倍稀释液（每200μL）至0.025微克/毫升。 </li></ol></li><li> CDC检测 <ol><li>收集从培养物中的Daudi和Daudi GFP +细胞和量化细胞活力（参见步骤1.2.1 - 1.2.3）。 </li><li>在无血清RPMI制备细胞悬浮液与4×10 5细胞/ mL。 </li><li>加将50μL细胞悬浮液加入到96孔圆锥形（V）底部微量培养板中的50μL每个参考或生物仿制mAb测试浓度。包括每个实验条件的重复。 </li><li>为阴性对照（ 即没有mAb）准备额外的孔，基础死亡控制（ 即在mAb存在下的热灭活NHSC）和染色阳性对照（ 即暴露于50μL70％EtOH的细胞）。 </li><li>在37℃，5％CO 2加湿气氛中孵育细胞20-30分钟。 </li><li>向每个孔中加入50μLNHSC（稀释1:2），并在37℃下在5％CO 2加湿气氛中孵育调理细胞2.5小时。在基础死亡控制井中使用热灭活的NHSC。 </li><li>在10℃下以400xg离心5分钟。丢弃上清液。 </li><li>通过加入150μLPBS洗涤细胞，并在400xg和10℃离心细胞悬浮液5分钟。迪清除上清液。 </li><li>用7-氨基生动生霉素（7-AAD）染色样品，如前所述7,8 。 </li><li>在同一天分析流式细胞仪上的细胞。 </li></ol></li><li> 数据采集 <ol><li>在流式细胞仪操作软件的工作表上打开两个点图。根据步骤1.3.1 - 1.3.3（ 图2A-B ）设置这些图。创建第三个图，这是G2群体上GFP与7-AAD的点阵图。 </li><li>使用Daudi细胞，Daudi GFP +细胞和死亡阳性对照来定义足够的FI限制（ 图2C ）。 </li><li>对于每个样品，测量7-AAD +靶细胞的百分比。从R2获取至少5,000个事件。 </li><li>通过从基础死亡对照中减去7-AAD +的百分比来计算具体的mAb诱导的细胞毒性，浓度的mAb（ 图2D ）。 </li></ol></li></ol>生物相似性分析<ol><li>将浓度和响应值输入图形软件。 </li><li>生成图形并计算非线性回归，具有以下考虑：i）使用mAb浓度的对数转换为“X”; ii）使用可变斜率数学模型（Y =最小响应+（最大响应 - 最小响应）/ 1 + 10 ^（（LogEC 50 -X）*山坡））;和iii）将底部值限制为零，因为已经减去了基础响应。 注意：预计有一个对称的S形曲线。 </li><li>使用F检验比较非线性拟合与全局拟合（许多图形软件程序包括此功能）。 注意：这样的测试建立为零假设，两个数据集的最大响应logEC 50和Hill斜率是相同的，这与预期生物学问题加以解决。 </li></ol>

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N. Salinas-Jazmín，González-González和SMPérez-Tapia是UDIBI的员工，负责为几家制药公司进行生物仿制研究。

治疗性单克隆抗体的专利期限正在促进生物仿制药的开发。因此，需要简单的方法来识别这些产品的临床相关活动的差异。 CD20 +培养细胞用于评估利妥昔单抗的两个关键功能特征：目标结合和CDC诱导。前一种活性需要通过mAb的Fab区域识别CD20，而后者主要取决于Fc区与其互补序列9的相互作用。因此，这些检测提供了一种链接mAbs的结构和功能特征的方法。 <p class="jove_con...

治疗性抗体是开发用于治疗不同病理学，包括癌症，自身免疫性和慢性疾病，神经系统疾病等的重组单克隆抗体（mAb）。目前，FDA批准了超过40种治疗性单克隆抗体，预计在接下来的几年内将有更多的药物进入市场。 利妥昔单抗是批准用于治疗CD20 + B细胞非霍奇金淋巴瘤（NHL），CD20 +滤泡性NHL，慢性淋巴细胞白血病和类风湿性关节炎2,3的高亲和力嵌合单克隆IgG1抗体。由利妥昔单抗对B细胞过度表达的CD20的识别诱导细胞凋亡;补体激活;和抗体依赖性细胞介导的细胞毒性（ADCC） 3 。该药物的专利于2013年和2016年在欧洲和美国期满， 分别。因此，制药公司全球正在开发的生物仿制药利妥昔单抗。与在其他任何药物用于人类消费，生物仿制药需要监管机构的批准。国际准则指出，对单克隆抗体，biosimilarity应通过比较理化性质，药代动力学，药效，以及新产品的参考4安全证明。 因此，在这样的比较所使用的方法必须评估mAb的结构和功能特征，特别是那些具有临床相关性。为此目的， 在体外测定法显示在体内实验几个优点（在Chapman 等人综述）。5：1） 体外研究是所提出的生物仿制药和参照产品之间的差异更加敏感; ⅱ） 在体内研究必须相关物种，这对于许多单克隆抗体是被执行非人灵长类和iii）由于作用机制，临床前毒理学和参考产品的临床效果是众所周知的，所以与生物仿制药的体内研究可能不提供额外的有用信息。因此，欧盟生物仿制药指南允许候选人基于稳健的体外数据进入临床试验6 。 在这里，我们提供两个快速，经济和简单的测定，评估使用CD20 +培养细胞的利妥昔单抗的生物活性。这些测定可以作为利妥昔单抗生物仿制药候选物的可比性研究的一部分。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>ATCC</td>
			<td>30-2001</td>
			<td>Modify the culture depending on the cell line</td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>0.4%, liquid, sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Daudi Burkitt&#39;s Lymphoma Cells</td>
			<td>ATCC</td>
			<td>CCL-213</td>
			<td>You can modify the cell line depending on the antibody of interest</td>
		</tr>
		<tr>
			<td>Fetal bovine serum(FBS)</td>
			<td>GIBCO</td>
			<td>16000-044</td>
			<td>You can modify the source of serum depending of requirements of the cell line</td>
		</tr>
		<tr>
			<td>Normal Human Serum Complement</td>
			<td>Quidel</td>
			<td>A113</td>
			<td>It is therefore appropriate for use in biocompatibility experiments including drug development, biomaterials testing and other applications</td>
		</tr>
		<tr>
			<td>7AA-D</td>
			<td>BDPharmigen</td>
			<td>559925</td>
			<td>You can use broad range of color options, compatible with most instrument configurations for to analyze viability.</td>
		</tr>
		<tr>
			<td>PECy5 Mouse Anti-human IgG</td>
			<td>BDPharmigen</td>
			<td>551497</td>
			<td>Change fluorochrome depending on the filter and laser of your flow cytometer.</td>
		</tr>
		<tr>
			<td>Human IgG Isotype Control</td>
			<td>ThermoFisher Scientific</td>
			<td>07-7102</td>
			<td>Change depending to mAb</td>
		</tr>
		<tr>
			<td>BDCytofix</td>
			<td>BDPharmigen</td>
			<td>554655</td>
			<td>Flow Cytometry Fixation Buffer (1-4% formaldehyde or paraformaldehyde )</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 10X (Phosphate buffer saline)</td>
			<td>GIBCO</td>
			<td>70011-044</td>
			<td>Phosphatebuffer without Ca2+/Mg2+ [137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4] and endotoxin free.</td>
		</tr>
		<tr>
			<td>Cell culture plates 96 well, V-bottom</td>
			<td>Corning</td>
			<td>29442-068</td>
			<td>12 x 75 mm round bottom test tubes or 96-well V- or U- bottom microtiter plates</td>
		</tr>
		<tr>
			<td>MabThera (Rituximab)</td>
			<td>Roche</td>
			<td>&#8212;</td>
			<td>Reference product</td>
		</tr>
		<tr>
			<td>Rituximab</td>
			<td>Indian</td>
			<td>&#8212;</td>
			<td>Biosimilar product</td>
		</tr>
		<tr>
			<td>15- or 50-mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430290 or 430052</td>
			<td>&#8212;</td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>Eppendorf</td>
			<td>&#8212;</td>
			<td>Multiple volume configurations are necessary</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf</td>
			<td>&#8212;</td>
			<td>Adjustable-volume pipettes are necessary</td>
		</tr>
		<tr>
			<td>Centrifuge 5430/ 5430R model</td>
			<td>Eppendorf</td>
			<td>&#8212;</td>
			<td>Refrigerated variable-speed centrifuge (4 to 25 &#176; C) with speeds ranging from 10 to 30,130 &#215; g</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Dickinson</td>
			<td>&#8212;</td>
			<td>BD FACSAria III or other flow cytometer</td>
		</tr>
		<tr>
			<td>Olympus optical and light microscope</td>
			<td>Olympus</td>
			<td>&#8212;</td>
			<td>To quantify and evaluate cell growth</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>SANYO</td>
			<td>&#8212;</td>
			<td>Incubatorfor temperature andCO2 control to culture cells</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet</td>
			<td>CHC BIOLUS</td>
			<td>&#8212;</td>
			<td>Biological safety cabinet that is used to protect the researcher, product and environment.</td>
		</tr>
	</tbody>
</table>

in vitro methods for comparing target binding and cdc induction between therapeutic antibodies: applications in biosimilarity analysis

治疗性单克隆抗体（mAb）与治疗不同病理学，包括癌症有关。制药公司开发生物仿制药是一个市场机会，但也是增加药物可及性并减少治疗相关成本的策略。这里详述的方案描述了利多西他单抗在Daudi细胞中的靶标结合和CDC诱导的评估。这两个功能需要抗体的不同结构区域，并且与由利妥昔单抗引起的临床效果相关。协议允许参考利妥昔单抗和市售的利妥昔单抗生物仿制药的对侧比较。评估的产品显示靶结合和CDC诱导的差异，表明存在潜在的物理化学差异，并强调需要分析这些差异在临床环境中的影响。这里报道的方法在体外构成简单且便宜/ em&gt;评估利妥昔单抗生物仿制药活性的模型。因此，它们在生物仿制发展过程中是有用的，也可用于生物仿制品的质量控制。此外，所提出的方法可以外推到其他治疗性mAbs。

使用上面描述的协议中，靶结合和参考利妥昔单抗的CDC诱导与产生的生物仿制药利妥昔单抗的和可商购于亚并行进行比较。 在Daudi细胞，两种mAb浓度依赖的方式（ 图1D）结合CD20。结合数据的非线性回归显示的0.978和0.848的R 2为参考和利妥昔单抗生物仿制药，分别（ 图1E）。浓度 - 响应曲线的统?...

Watch this Scientific Journal Video about In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis at JoVE.com

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

该协议描述了利妥昔单抗的两个关键功能特征的体外比较：靶结合和补体依赖性细胞毒性（CDC）诱导。该方法用于参考利妥昔单抗和利妥昔单抗生物仿制药之间的对侧比较。这些测定可以在生物仿制发展过程中使用或作为其生产中的质量控制。

体外方法比较标的结合和CDC诱导治疗性抗体之间：应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

The overall goal of this procedure is to compare two key functional characteristics between therapeutic antibodies, target binding, and CDC induction during their biosimilar development, or as a quality control, during their production.These methods can help answer our key questions about how to assess functional differences between therapeutic antibodies that bind the same target protein.These techniques are quick, simple, and inexpensive, allowing for day execution in most laboratories.Demonstrating the procedures will be Edith Gonz

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Research

JoVE Journal

Immunology and Infection

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

17.6K 次观看.  National Polytechnic Institute (IPN), University of Mexico (UNAM). The overall goal of this procedure is to compare two key functional characteristics between therapeutic antibodies, target binding, and CDC induction during their biosimilar development, or as a quality control, during their production.These methods can help answer our key questions about how to assess functional differences between therapeutic antibodies that bind the same target protein.These techniques are quick, simple, and inexpensive, allowing for day execution in most laboratories.Demo...

视频: In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

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 ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

一个<em&gt;体外</em&gt; Caseum结合测定，预测药物在渗透结核病变

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...