支持这项研究是由来自西肯塔基大学学院留学基金管理委员会的新教师津贴。

1。手指沾采血<ol><li>休息时通常采取的收集全血为基准测量（通常是10-15分钟坐下休息），期间或之后立即行使回合，和整个运动后期间（一般1-3小时以下停止练习）。 </li><li>使用通用的预防措施，首先消毒，用70％异丙醇的指尖。 </li><li>下一步，穿刺网站使用自动柳叶刀或类似设备。虽然我们使用30号的柳叶刀为主题的舒适，较大的针头可利用。 </li><li>擦去第一滴血，然后收集到毛细管或锂肝素治疗，以防止凝血microvettes的血液。 </li></ol> 2。细胞表型和细胞凋亡染色<ol><li>加入10μL肝素化全血250μL结合缓冲液的滴度抗体面板（见表1原理）。我们已经成功地使用1:20稀释因子，但每个实验室应滴定试剂，以确定最好的工作方案。 表1。运动诱导细胞凋亡的白细胞亚群测定抗体面板。早期凋亡Annexin V的表达，而晚期凋亡Annexin V和7 - AAD双正面标签中定义的界定。坏死细胞的膜联蛋白V -和7 - AAD +细胞只。 <table border="1"><tbody><tr><td>管1 </td><td>管2 </td><td>管3 </td><td>管4 </td></tr><tr><td>细胞管</td><td>膜联蛋白V - FITC </td><td>膜联蛋白V - FITC </td><td>膜联蛋白V - FITC </td></tr><tr><td></td><td>抗人CD4 - 体育</td><td>抗人CD8 + - PE </td><td>抗人CD19 - PE </td></tr><tr><td></td><td> 7 - AAD </td><td> 7 - AAD </td><td> 7 - AAD </td></tr><tr><td></td><td>抗人CD45RA - APC </td><td>抗人CD45RA - APC </td><td></td></tr></tbody></table> 7 - AAD = 7 - 氨基放线菌素D，APC =变藻蓝蛋白，CD4 +辅助性T淋巴细胞，CD8 +抑制性/细胞毒性T淋巴细胞，CD19 + = B淋巴细胞，CD45RA + =幼稚细胞亚群，FITC异硫氰酸荧光素，PE =藻红蛋白。 </li><li>在室温下在黑暗中孵育30分钟。 </li><li>离心机在1075 XG为5-10分钟。 </li><li>倒出，加300μL红血细胞裂解液，彻底旋涡。 </li><li>在室温下孵育15分钟。 </li><li>添加300μLPBS停止红细胞裂解反应。 </li><li>离心机在1075 XG为5-10分钟。 </li><li>倒出，机架，并添加50μL结合缓冲液。 </li><li>流式细胞仪分析。 </li></ol>这是建议，至少以下控制利用：仅管作为一个背景自体荧光检测阴性对照的一个细胞，每一个人荧光管设置在流式细胞仪的初步赔偿。此外，我们已经使用了补偿标准珠，成功地建立我们的实验。

<ol>
	<li>Simpson, R. J., Florida-James, G. D., Whyte, G. P., Black, J. R., Ross, J. A., Guy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+does+not+contribute+to+the+blood+lymphocytopenia+observed+after+intensive+and+downhill+treadmill+running+in+humans.">Apoptosis does not contribute to the blood lymphocytopenia observed after intensive and downhill treadmill running in humans.</a> Res. Sports Med. 15, 157-174 (2007).</li><li>Steensberg, A., Morrow, J., Toft, A. D., Bruunsgaard, H., Pedersen, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+exercise,+lymphocyte+apoptosis+and+F2-isoprostanes.">Prolonged exercise, lymphocyte apoptosis and F2-isoprostanes.</a> Eur. J. Appl. Physiol. 87, 38-42 (2002).</li><li>Mooren, F. C., Bloming, D., Lechtermann, A., Lerch, M. M., V&#246;lker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+apoptosis+after+exhaustive+and+moderate+exercise.">Lymphocyte apoptosis after exhaustive and moderate exercise.</a> J. Appl. Physiol. 93, 147-153 (2002).</li><li>Peters, E. M., Eden, M. V. a. n., Tyler, N., Ramautar, A., Chutergoon, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+exercise+does+not+cause+lymphocyte+DNA+damage+or+increased+apoptosis+in+well-trained+endurance+athletes.">Prolonged exercise does not cause lymphocyte DNA damage or increased apoptosis in well-trained endurance athletes.</a> Eur. J. Appl. Physiol. 98, 124-131 (2006).</li><li>Wang, J. -. S., Huang, Y. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+intensity+on+lymphocyte+apoptosis+induced+by+oxidative+stress+in+men.">Effects of exercise intensity on lymphocyte apoptosis induced by oxidative stress in men.</a> Eur. J. Appl. Physiol. 95, 290-291 (2005).</li><li>Mooren, F. C., Lechtermann, A., V&#246;lker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-induced+apoptosis+of+lymphocytes+depends+on+training+status.">Exercise-induced apoptosis of lymphocytes depends on training status.</a> Med. Sci. Sports Exerc. 36, 1476-1483 (2004).</li><li>Navalta, J. W., Sedlock, D. A., Park, K. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+treatment+influences+the+yield+of+apoptotic+lymphocytes+after+maximal+exercise.">Blood treatment influences the yield of apoptotic lymphocytes after maximal exercise.</a> Med. Sci. Sports Exerc. 37, 369-373 (2005).</li></ol>

一种微创的样品收集过程和随后的分析表明运动诱导淋巴细胞凋亡的早期和晚期阶段的分析。它也可以明确地排除那些坏死，并没有凋亡的细胞，这是。此过程克服的弊端，从肘地区侵入静脉穿刺，在多个时间点之前相关的不适，期间和运动后即刻或安全行使回合期间导管。在调查中，这类程序通常需要放血，并非所有个人的专门培训。此外，受招聘和保留使用此过程预计将增强，从而增加了发?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Flow Cytometry Binding Buffer</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>00-4222-26</td>
<td>&nbsp;</td>
<tr>
<td>RBC Lysis Buffer</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>00-4333-57</td>
<td>&nbsp;</td>
<tr>
<td>Lancet Device</td>
<td>Accu Check Soft Touch</td>
<td>Roche Diagnostics Corp.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Capillary tubes</td>
<td>Heparinized Capillary Tubes</td>
<td>Chase Scientific Glass, Inc.</td>
<td>501</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>GmC Lab</td>
<td>Gilson</td>
<td>PMS-880</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-FITC Conjugated</td>
<td>&nbsp;</td>
<td>Biovision Inc.</td>
<td>K201-400-1</td>
<td>&nbsp;</td>
<tr>
<td>CD4-PE Conjugated</td>
<td>Anti-human, clone RPA-T4</td>
<td>eBioscience</td>
<td>12-0049-42</td>
<td>&nbsp;</td>
<tr>
<td>CD8-PE Conjugated</td>
<td>Anti-human, clone OKT8</td>
<td>eBioscience</td>
<td>12-0086-73</td>
<td>&nbsp;</td>
<tr>
<td>CD19-PE Conjugated</td>
<td>Anti-human, clone HIB19</td>
<td>eBioscience</td>
<td>12-0199-42</td>
<td>&nbsp;</td>
<tr>
<td>7-AAD Viability Staining Solution</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>00-6993-50</td>
<td>&nbsp;</td>
<tr>
<td>CD45RA-APC Conjugated</td>
<td>Anti-human, clone HI100</td>
<td>eBioscience</td>
<td>17-0458-42</td>
<td>&nbsp;</td>
<tr>
<td>Flow cytometer</td>
<td>C6</td>
<td>Accuri</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

finger-stick blood sampling methodology for the determination of exercise-induced lymphocyte apoptosis

运动能诱导免疫细胞凋亡是一种生理刺激。种种限制，迄今为止，已确定这种现象的测量，尤其是有关的细胞死亡的评估之前，隔离和治疗血液样本所需的时间。正因为如此，它是难以确定是否增加免疫细胞凋亡的报道可以促进运动对系统的实际效果，或反映了必要的时间和处理，最终获得此测量。在这篇文章中，我们展示了运动诱导淋巴细胞凋亡分析一个快速和微创手术。与其他技术不同的是，全血立即被添加到抗体面板后，获得一个样品。潜伏期之后，红血细胞裂解样品准备进行分析。手指棒抽样程序的使用减少了所需的血​​液量，最大限度地减少科目的不适。

Watch this Scientific Journal Video about Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis at JoVE.com

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

锻炼是能诱导免疫细胞凋亡。有各种测量的局限性，特别是有关的评估之前，隔离和治疗血液样本所需的时间。证明是一个快速运动诱导淋巴细胞凋亡的分析和微创手术。

手指棒运动诱导淋巴细胞凋亡的测定血液抽样方法

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

finger-stick-blood-sampling-methodology-for-determination-exercise

Research

JoVE Journal

Immunology and Infection

12.1K 次观看.  Western Kentucky University. 锻炼是能诱导免疫细胞凋亡。有各种测量的局限性，特别是有关的评估之前，隔离和治疗血液样本所需的时间。证明是一个快速运动诱导淋巴细胞凋亡的分析和微创手术。

视频: Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

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 ...

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

羧基荧光素二醋酸琥珀酰亚胺酯（CFSE）用于监视淋巴细胞增殖

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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 ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...