研究已经得到了华莱士阁下科尔特基金会早在转化研究事业奖。采血的帮助下添安德鲁斯，小儿血液/肿瘤，。也感谢萨姆豪森博士，流式细胞仪的核心，詹姆斯格雷厄姆布朗癌症中心，运行流式细胞仪检测样品的帮助。

第1部分。微流控设备制造创建一个微流体裂解装置的硅母模使用标准光刻技术与设备，在路易斯维尔洁净室大学[1]。 AutoCAD的（欧特克公司，圣拉菲尔，CA）是用来生成一个透明的面具（Fineline成像，科罗拉多州的斯普林斯，CO），创建渠道的负面副本。从硅掌握使用弹性体聚二甲基硅氧烷（PDMS;道康宁，美联，MI）的软光刻技术的模具制造微流体裂解装置。弹性体与复制的渠道被释放，并用20号针头（小零件，美丽华，佛罗里达州。）信道接入孔打孔。 PDMS的晶圆是不可逆转的保税通过氧等离子体载玻片和实验测试。 第2部分。运行裂解装置的议定书“ 微流体装置已经制造和测试后，红细胞裂解试验准备开始。以下部分详细说明协议的微流体装置。 2.1样品采集<ol><li> 4毫升的血液样本收集从肘正中静脉，肝素作为抗凝血剂2毫升绿色顶级vaccutainers前对病人的前臂静脉。 </li><li>放弃第2毫升的管，因为它包含脱落的成熟内皮细胞（误报）。 </li><li>重悬第二管，肝素与血液混合，向​​上和向下转动;立即保存在冰上。 </li><li> 1毫升血液转移到1.5 mL Eppendorf管。立即进程的血液样本（收集一小时内）。 </li></ol> 2.2微流体装置，用PBS总理<ol><li>获取粘接到玻璃微流体装置。按合适的访问油管（小零件，美丽华，佛罗里达州），在入口和出口（图1）的直径略大。 </li><li> 30号注射器针头（小零件，美丽华，佛罗里达州）连接到每个管的进气口，提供宏观到微观的接口。 </li><li>填写与1X PBS 1 ml注射器，并确保获得摆脱气泡弹注射器针头。 PBS装1 ml注射器连接入口1的微流体装置。推注射器含有1X PBS轻轻用手，直到溶液流进2，然后立即钳与一个标准的Office活页夹夹入口2 </li><li>继续推动1X PBS液，直到达到微流体设备的出油口。一旦解决方案流出出水口，出口管钳与另一粘结剂剪辑。 </li><li>继续推进1 ml注射器，直到1X PBS流进3，和与其他Office活页夹夹钳入口3端口。微流体装置现在已经完全做好准备。 </li></ol><img src="/files/ftp_upload/1656/1656fig1.jpg" alt="图1" /> 图1显示各自的解决方案和插座入口装置的示意图。 2.3制备注射泵和解决方案<ol><li>要校准泵，遵循以下步骤： <ol class="lalpha"><li>对于大哈佛注射泵（哈佛大学，PHD 22/2000部分＃702000）：22.5毫米，流速600μL/ min的设置直径配置。 </li><li>对于小哈佛注射泵（哈佛，碧加上部分＃702213）：设置直径配置4.61毫米，流速20μL/分钟。 </li></ol></li><li>填写一个30毫升注射器，用无菌去离子水。这可以通过直接退出的解决方案，从50 ml锥形管。 （标签注射器） </li><li> 2X磷酸盐缓冲液（PBS），2％多聚甲醛（PFA）在第9步中所述的程序，填写第二个30 ml注射器。 （标签注射器） </li><li>填写与1X PBS 1 ml注射器从15 ml锥形管中的分装，使用在第9步中所述的程序。 </li><li>将30 ml去离子水入口2注射器和2X PBS 30毫升注射器入口3（确保你唐吨陷阱气泡在管或微流体装置）。拆下进气口2和3和出口管钳。 1X PBS 1 ml注射器连接到进气口1。避免引入气泡填充液体的注射器针头，注射器连接，并轻弹注射器针头摆脱泡沫。 </li></ol> 2.4红细胞裂解<ol><li>注射器上设置适当的哈佛泵概述如下： <ol class="lalpha"><li>大哈佛注射泵设置的两个30毫升注射器（一个含无菌，去离子水和其他与2X PBS，2％的煤灰）。 </li><li>设置1毫升（含1 × PBS）上的小哈佛注射泵的注射器。 </li><li>在废物收集商将出口管（50 ml锥形管，标记瓦特阿斯泰）。 </li></ol></li><li>打开大哈佛注射泵（用30毫升注射器），并让它运行一分钟。 （确保没有气泡和没有在设备或管路中泄漏）。 </li><li>打开小哈佛注射泵上，并让它运行额外的分钟。 （确保没有气泡和没有在设备或管路中泄漏）。停止泵。微流体设备现在已经可以使用。 </li><li>删除小哈佛注射泵包含1X PBS 1 ml注射器。 </li><li>取得血液样本后，填写用0.05毫升无菌1X PBS 1 ml注射器，没有捕获任何气泡。接下来，填充注射器0.5毫升的血液从Eppendorf管中获得。 （1 ml注射器将包含一个最终成交量为0.55毫升的血液和1X PBS缓冲液。） 注 ：保持注射器垂直却使避免0.05毫升PBS的血液混合。 </li><li>连接入口1含有血液的注射器和仔细装入注射器上的小哈佛注射泵（注射器装到泵垂直）（避免推到设备的血液通过管子）。 </li><li>从废液管出口管取出，放在它的位置在一个干净的50 mL离心管中，并设置它的冰桶。 </li><li>第一大哈佛注射泵开关，并让它运行1分钟。开始收集样品从插座到50 mL离心管。 </li><li>开关上的小哈佛注射泵。 </li><li>一旦在1毫升注射器的血液样本已完全通过设备前往，停止这两个泵。这大约需要20分钟。 </li><li>离心5分钟收集到的样品在室温制动关闭在350 XG。 </li><li>去除上清液放置在对面的白色颗粒枪头。删除尽可能多的可能，尤其是红色的细胞碎片上清，不扰民的白色颗粒。 </li><li>向上和向下吹打重悬在1毫升流缓冲区（1X PBS，1％BSA牛血清白蛋白]，2％PFA）的样品。转移到1.5 mL Eppendorf管的样品。 </li><li>离心5分钟在室温下在4 XG所收集的样本。使用相同的精度，前去除上清。 </li><li>重悬在1毫升流缓冲震荡的样品。 </li></ol> 2.5流式细胞仪分析的准备工作<ol><li>使用流式细胞仪分析样品，加100μL的样品，以流式细胞仪检测管。 </li><li>添加指定的抗体（确保正确的浓度）的样品100μL。 </li><li>允许样品孵育40 C. 30分钟</li><li>洗两次流的缓冲区之前，流式细胞仪。清洗步骤包括添加250流缓冲液，振荡，离心5分钟，在350 XG，并移除上清液流式细胞仪检测管流体转。 </li><li> 250流缓冲液重悬沉淀。样品是准备通过流式细胞仪分析。 </li></ol>代表性的成果通过微裂解装置和后处理协议的全血已运行，消除红细胞和有核细胞（CNCS）丰富循环。理想的结果将产生一个单独的数控人口的清洁流式细胞仪散点图。在下面的图2和3显示的是全血样本的散点图比较无红细胞裂解红细胞裂解与微流控协议前向散射（FSC）与侧散射（SSC），光轴。由此可以看出，微流体富集协议是必要的，以获得一个干净的流式细胞仪散点图的。 <img src="/files/ftp_upload/1656/1656fig2.jpg" alt="图2" /> 图2流式细胞仪检测全血样品的分散没有与FSC和SSC轴红细胞裂解情节。 <img src="/files/ftp_upload/1656/1656fig3.jpg" alt="图3" /> 图3。核细胞循环与微流体溶解红细胞人口与FSC和SSC轴标记流式细胞仪散点图。 1区（红色）代表区域2（绿色）代表单核细胞，淋巴细胞，3区（蓝色）代表粒，4区（紫色）尚未特点。 协议中的吸管去除红细胞碎片也必要时重要。在下面的图4是散点图呈现出红细胞碎片的盈余。虽然不是混浊的散点图，如图2，必须考虑到由于红细胞​​碎片在数据分析中的添加事件。 <img src="/files/ftp_upload/1656/1656fig4.jpg" alt="图4" /> 图4。流式细胞仪散点图描绘红细胞碎片，被视为一个集群5区（青色）的事件。 为某些抗体表型标记染色细胞也会产生特征的结果。在下面的数字是3个不同的白细胞人口渍：抗体表型淋巴细胞，单核细胞，和粒细胞。图5描述了绘制与轴CD3和CD4淋巴细胞人口。单核细胞和粒细胞作为FSC对CD14和CD66b轴在图6-7所示，分别为。 <img src="/files/ftp_upload/1656/1656fig5.jpg" alt="图5" /> 图5。流式细胞仪散点图描绘淋巴细胞CD3和CD4轴。 <img src="/files/ftp_upload/1656/1656fig6.jpg" alt="图6" /> 图6描绘与FSC和CD14轴的单核细胞流式细胞仪散点图。 <img src="/files/ftp_upload/1656/1656fig7.jpg" alt="图7" /> 图7。流式细胞仪散点图描述与FSC和CD66b轴粒。

<ol>
	<li>White, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+application+of+microfluidic+leukocyte+enrichment+protocol+in+mild+phenotype+sickle+cell+disease+(SCD.">Clinical application of microfluidic leukocyte enrichment protocol in mild phenotype sickle cell disease (SCD.</a> Biomedical Microdevices. 11 (2), 477-483 (2009).</li><li>Sethu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Flow+Microfluidic+Device+for+Rapid+Erythrocyte+Lysis.">Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis.</a> Anal. Chem. 76 (21), 6247-6253 (2004).</li></ol>

一个自动化微流体血液溶解协议的报道。值得一提的是在协议的关键步骤。在第P.2.1，重要的是要丢弃的血液小瓶样品中含有成熟的内皮细胞脱落，误报收集。另外，还要确保运行一小时内收集的血液样本。底漆第P.2.2微流体装置，它是重要的，初步摆脱了气泡。此外，应避免引入气泡时附加新的整个协议的针头注射器。 P.2.4节血1 ml注射器灌装时，一定要记住先添加1X PBS和血液，所有同时保持垂?...

automated microfluidic blood lysis protocol for enrichment of circulating nucleated cells

一个自动化微流体血液裂解装置的协议，在本报告中详细介绍。循环有核细胞（CNCS），包括白细胞和内皮细胞，提供一个理想的平台，为个人的免疫状态的更新状态。微流体协议允许无选择性的细胞损失和样品制备变性，由于用户介导的步骤浓缩的CNC。简单地说，协议包括设备制造，样品的采集，设备安装，并通过微腔血。整个器件内的血液迅速去离子水混合，在约10秒50微米× 150微米的微流体通道。在这个时间跨度红细胞裂解由于低渗条件。通道底部的人字形结构，确保充分混合细胞暴露为一个恒定的环境。剩余的单元格返回等渗条件下，在设备的出口，用2％多聚甲醛固定，离心分离CNC的红细胞碎片，并在流缓冲暂停染色和流式细胞仪分析。结果表明清洁流式细胞仪散点图数控保存种群。这种设备和协议的意义数控人口分析疾病的发病机制的研究和理解。因此，自动化，实效性，简单的微流控协议证明。

Watch this Scientific Journal Video about Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells at JoVE.com

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

发展循环通过红细胞裂解，以确保高质量的样品无细胞损失隔离外周血有核细胞富集自动化微流体装置。

微流控自动化血液循环核细胞富集的裂解协议

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

automated-microfluidic-blood-lysis-protocol-for-enrichment

Research

JoVE Journal

Biology

12.3K 次观看.  University of Louisville. 发展循环通过红细胞裂解，以确保高质量的样品无细胞损失隔离外周血有核细胞富集自动化微流体装置。

视频: Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

NK细胞与干细胞技术的RosetteSep套件的人体血液的富集

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

科研

生物学

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

cd4+ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a problem for physicians and researchers. This manuscript describes a simple and effective method to monitor immune rejection in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models. Circulating DNA is a potentially non-invasive biomarker for organ damage. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection by quantitative real-time PCR (qPCR). In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Species-specific primers were then used to quantify cpsDNA by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models. The value of this method suggests that it can be used as a simple, convenient, low cost, and less invasive method to monitor the immune rejection of xenotransplantation.

In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Using species-specific primers, cpsDNA was quantified by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models.

异种移植模型中循环猪特异性DNA的定量

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a ...