所AdvanDx赞助的研究。

1。标本采集和准备<ol><li>收集怀疑败血症患者的静脉血，放入两个BACTEC（BD，火花，MD）血培养瓶，一个有氧之一厌氧一起组成一个血培养集，。 </li><li>广场瓶BACTEC 9240仪器35 ° C。 </li><li>孵育直到仪器触发报警，由于微生物的生长，血培养瓶。 </li><li>取出一瓶血培养仪。 </li></ol> 2。试剂的制备染色前<ol><li>准备加入4毫升新鲜去离子水或蒸馏水240毫升直接染色菜60X洗净解决方案的洗涤液。 </li><li>放置在55℃水浴染色菜温暖​​。 </li><li>存储其余集中在2-8 ° C。 </li><li>从冰箱中取出安装介质和温暖的室温。 </li></ol> 3。革兰氏染色<ol><li>轻轻摇动血培养瓶。 </li><li>使用针头和注射器或传代仪器，删除从瓶子在无菌螺丝上限管血（约5毫升）。 </li><li>使用无菌吸管，将一个血一个玻璃显微镜玻片上的大幅下降。 </li><li>空气干燥的幻灯片和甲醇固定为10秒。 </li><li>允许幻灯片空气干燥。 </li><li>进行革兰氏染色血膜，以确定血液中培养瓶中生长着的有机体的形态。 </li><li>使用油浸镜头，查看幻灯片。在对和观察到的短链的革兰氏染色的革兰氏阳性球菌是最肠球菌种。 </li><li>这种革兰氏染色的结果，那么驱动器选择适当的PNA FISH探针试剂盒，用于细菌鉴定。这血培养将被染色使用的大肠杆菌粪 / OE PNA鱼染色。 </li></ol> 4。 PNA FISH染色<ol><li>混合管中的血液轻轻纷飞前涂抹准备。 </li><li>放置一个固定的解决方案上的PNA FISH显微镜幻灯片以及下降。 </li><li>从管与血培养阳性10μL或一个小水滴转移到固定的解决方案，并轻轻混合乳化。 </li><li>修复加热20分钟涂片。在55 ° C在加热板。 </li></ol> 5。质量控制材料一个正极和一个负极的质量控制幻灯片，必须进行测试，每批染色幻灯片。 <ol><li>使用E。粪 / OE控制幻灯片从AdvanDx购买用于此目的，或准备从液体培养 E.参考株细胞涂片粪肠球菌和大肠杆菌作为阳性对照，无论是在单独的幻灯片或上一张幻灯片和菌金黄色葡萄球菌或链球菌为阴性对照材料混合肠球菌。 </li></ol> QC成果应该能够监测相应的检测条件，特别是那些影响杂交紧缩和细胞壁渗透，因为巴勒斯坦民族权力机构的方法，旨在优化细胞壁渗透 6。杂交<ol><li>添加E 的一滴粪 / OE巴勒斯坦民族权力机构与涂片的显微镜幻灯片以及。 </li><li>加盖玻片。避免产生气泡。 </li><li>孵育30 ± 5分钟。在55 ± 1 ° C的加热板</li><li>以下孵化的地方的幻灯片，在幻灯片的载体</li></ol> 7。严格的清洗<ol><li>在预热的清洗液浸泡滑动载体，在55 ° C和小心取出盖玻片。通常情况下，盖玻片，轻轻搅拌清洗液中的幻灯片滑出。有时，必须轻轻推盖玻片，用镊子。 </li><li>孵育在洗涤液30 ± 5分钟。在55 ± 1 ° C。 </li><li>从洗涤液中取出的幻灯片</li><li>允许在空气干燥的幻灯片</li></ol> 8。安装<ol><li>添加一个安装介质下降到每张幻灯片</li><li>加盖玻片。避免产生气泡。 </li><li>在2小时内的荧光显微镜检查的幻灯片。 </li><li>不要让幻灯片阳光直接照射或其他强光源，因为这可能导致荧光猝灭。 </li></ol> 9。结果分析必须配备AdvanDx双带通滤波器和一个60X或100X油客观，在荧光显微镜用于幻灯片检查。质量控制幻灯片，应首先检查确认，杂交确实发生。大肠杆菌粪应显示为明亮的绿色荧光球菌在多个领域的观点和屎肠球菌会出现明亮的红球菌。非肠球菌控制幻灯片应该出现nonfluorescent的。控制系统确认后，可以检查病人的幻灯片。起初，血液过滤米会出现偏红，但鲜艳的红色和绿色球菌会相当明显。 <img src="/files/ftp_upload/2616/2616fig1.jpg" alt="图1" /> 代表性的例子的绿色阳性E. 图1。 粪肠球菌 （左），红积极屎肠球菌（中）和负（右）的测试结果。 10。代表性的成果三个机构，包括在一个多中心临床试验，评估这个PNA FISH染色和比较一个协议，以2.5小时缩短1.5小时的协议，刚刚被审查。共有152常规革兰氏阳性球菌对和链条（GPC）阳性血培养瓶被列入研究。协议修改后的结果是100％（152分之152）和E.原检测程序粪 / OE PNA鱼:41/41 E。粪肠球菌 ; 33/33肠球菌等78分之78其他的GPC（见表1）。此染色法在这项临床试验（表2）精美的敏感性和特异性。 <table border="1"><tbody><tr><td></td><td> 常规方法 </td><td> 粪肠球菌 / OE PNA FISH标准程序 </td><td> 粪肠球菌 / OE PNA FISH简短过程 </td></tr><tr><td> 粪肠球菌 </td><td> 41 </td><td> 41（绿正面） </td><td> 41（绿正面） </td></tr><tr><td> 屎肠球菌 </td><td> 27 </td><td> 27（红色正） </td><td> 27（红色正） </td></tr><tr><td> E. casseliflavus </td><td> 2 </td><td> 2（红色正） </td><td> 2（红色正） </td></tr><tr><td> E.鹑鸡 </td><td> 2 </td><td> 2（红色正） </td><td> 2（红色正） </td></tr><tr><td> 其他肠球菌。 </td><td> 2 </td><td> 2（红色正） </td><td> 2（红色正） </td></tr><tr><td> 肺炎链球菌 </td><td> 17 </td><td> 17（负） </td><td> 17（负） </td></tr><tr><td> 草绿色链球菌 </td><td> 33 </td><td> 33（负） </td><td> 33（负） </td></tr><tr><td> S. mitis </td><td> 1 </td><td> 1（负） </td><td> 1（负） </td></tr><tr><td> 化脓性链球菌 </td><td> 3 </td><td> 3（负） </td><td> 3（负） </td></tr><tr><td> 南牛 </td><td> 2 </td><td> 2（负） </td><td> 2（负） </td></tr><tr><td> 南唾液 </td><td> 1 </td><td> 1（负） </td><td> 1（负） </td></tr><tr><td> 南血统 </td><td> 2 </td><td> 2（负） </td><td> 2（负） </td></tr><tr><td> 南agalagtae </td><td> 7 </td><td> 7（负） </td><td> 7（负） </td></tr><tr><td> 其他链球菌。 </td><td> 7 </td><td> 7（负） </td><td> 7（负） </td></tr><tr><td> Abiotrophia SPP。 </td><td> 3 </td><td> 3（负） </td><td> 3（负） </td></tr><tr><td> Peptostrepococcus SPP。 </td><td> 2 </td><td> 2（负） </td><td> 2（负） </td></tr><tr><td>共有</td><td> 152 </td><td> 152分之152 100％协议</td><td> 152分之152 100％协议</td></tr></tbody></table> 表1。上市细菌测试，使用标准（2.5hr）和快速（1.5小时）协议。 <table border="1"><tbody><tr><td> 灵敏度E.faecalis </td><td> 灵敏度其他肠球菌。 </td><td> 特异性 </td></tr><tr><td> 100％（41/41） 95％CI（93.0-100） </td><td> 100％（33/33）33/33 95％CI（91.3-100） </td><td> 100％（七十八分之七十八） 95％CI（96.2-100） </td></tr></tbody></table> 表2。1.5小时PNA FISH协议的灵敏度和特异性。

<ol>
	<li>Oliveira, K., Chapin, K. C., Musgnug, M. C., Haase, G., Weber-Heynemann, J., DeGirolami, P. C., Dakos, J., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures+by+dual+color+fluorescence+in+situ+hybridization+using+PNA+probes.">Rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures by dual color fluorescence in situ hybridization using PNA probes.</a> , (2002).</li><li>Chapin, K. C., Musgnug, M. C., Oliveira, K., DeGirolami, P. C., Dakos, J., Johansen, J. T., Procop, G. W., Wilson, D., Padilla, E., Gonzalez, V., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+E.+faecalis+PNA+FISH+for+rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures.">Multicenter evaluation of E. faecalis PNA FISH for rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures.</a> , (2003).</li><li>Rausei, V., Youssef, E., Morgan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+Staphylococcus+aureus,+Enterococcus+faecalis,+and+Candida+albicans+from+blood+cultures+using+fluorescence+in+situ+hybridization+with+nucleic+acid+probes.">Direct identification of Staphylococcus aureus, Enterococcus faecalis, and Candida albicans from blood cultures using fluorescence in situ hybridization with nucleic acid probes.</a> , (2005).</li><li>Novak-Weekley, S. M., LaForga, M., Carey, L., Frazier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+S.+aureus+PNA+FISH,+E.+faecalis+PNA+FISH+&#38;+C.+albicans+PNA+FISH+for+Rapid+Identification+of+Positive+Blood+Culture+Bottles.">Validation of S. aureus PNA FISH, E. faecalis PNA FISH &#38; C. albicans PNA FISH for Rapid Identification of Positive Blood Culture Bottles.</a> , (2006).</li><li>Johnson, J. K., Roberts, A. A., Forrest, G. N., Lincalis, D. P., Venezia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Enterococcus+Species+in+Positive+Blood+Cultures+with+Therapeutic+Implications.">Rapid Identification of Enterococcus Species in Positive Blood Cultures with Therapeutic Implications.</a> , (2006).</li></ol>

生产这种视频文章是由AdvanDx赞助。本杰明水晶AdvanDx，本文中使用的工具和试剂的雇员。

的大肠杆菌肠球菌粪 / OE PNA FISH检测可以提供鉴定的标准培养方法相比，提前2-3天。缩短检测程序（1.5 hourr）提供了快速识别抗菌治疗和患者预后的一个更好的方法可以使用​​。福雷斯特， 等。（6）支持的一项研究。微生物实验室在2005年开始连续两年确定在双和链增长由传统的微生物学方法在血培养瓶，并于2006 年加入大肠杆菌革兰氏阳性球菌粪 / OE PNA鱼。此外治疗机构抗菌队（AMT）开发的算法，有效地使用PNA FISH及时实验室生成的数据。主要成果评估时间从血培养提请实施有效的抗菌治疗前，后进入实验室工作流程PNA FISH提起。严重性疾病，病人的位置和经验性抗菌治疗。共有224医院获得性肠球菌菌血症患者进行了评价与129前干预期间，在PNA FISH期间95。 PNA FISH鉴定大肠杆菌粪 3天前比传统文化（1.1 - 4.1天，p &lt;0.001）。 PNA FISH确定屎肠球菌中位数为2.3天（1.1比3.4天，P &lt;0.001），与时间统计学显着减少，发起有效的治疗方法（1.3 - 3.1天，p &lt;0.001）和30天的下降死亡率（26％比45％，P = 0.04）。这个小组的结论是，在大肠杆菌在适当的经验性抗菌治疗早期开始收购的粪大肠杆菌菌血症患者与医院粪/ OE PNA FISH检测结果结合AMT处理算法。 <ol><li>不应该以任何方式修改后到期日期印在标签和试剂，试剂，不得使用。他们应该留在冰箱在不使用时，或他们可能会失去效力。 </li><li>需要混合采样前血培养瓶。 </li><li>重要的是不允许的滴管瓶端触摸血涂片，因为这可能会导致材料幻灯片之间的交叉污染，或造成污染的试剂。 </li><li>确保保持在一个恒定的55℃水浴加热块</li><li>不要使用其他过滤器比双带通滤波器或颜色不会清楚看到。 </li><li>不要使用比显微镜幻灯片或杂交不会发生其他的显微镜幻灯片。 </li><li>重要的是显微镜的正常运作。确保显微镜灯泡使用时间内批准数。 </li></ol><ul><li>一些罕见​​的发生肠球菌物种无法检测到的PNA探针杂交肠球菌的其他品种，如E. asinii，E.dispar，大肠杆菌haemoperoxidus，大肠杆菌cecorum，大肠杆菌columbae，大肠杆菌saccharolyticus，孤东和E， 。硫磺</li><li> E. moraviensis被鉴定为E。由于序列相似的粪。 </li><li>一些链球菌 anginosus菌株产生积极虚假的绿色荧光，由于序列相似性。 </li><li> VersaTREK血培养瓶在临床研究中未评估。性能检测大肠杆菌粪肠球菌和其他肠球菌菌属。与VersaTREK血培养瓶是未知之数。 </li><li>如果是采用双带通滤波器，而不是一个标准的FITC标记的过滤器可能会出现假阳性的绿色自发荧光。 </li><li>假阴性结果可能会经常发生由于混合生长，或由于检测技术中的错误。 </li><li>所用仪器的类型和条件会影响所获得的图像的视觉外观。荧光可能varydue类型的显微镜，光源和rRNA基因在细胞水平</li><li>固体培养基上的分离是需要区分与其他生物的混合的生长，并产生了负面的结果，以确定血培养阳性。 </li><li>比血培养标本，该产品还没有得到验证。 </li></ul>

试剂 粪肠球菌 / OE PNA FISH以下套件的组件包括: <ul><li>固定解决方案固定解决方案3毫升磷酸盐缓冲生理盐水用洗涤剂。 </li><li> 粪肠球菌 / OE巴勒斯坦民族权力机构大肠杆菌粪 / OE PNA 1.5毫升PNA杂交探针。解决方案。含有30％甲酰胺。 </li><li> 60X清洗液60X洗涤溶液50 ml的Tris缓冲液用洗涤剂</li><li>安装介质安装甘油液3毫升漂白抑制剂</li><li>质量控制幻灯片</li><li> <a href="http://www.advandx.com">AdvanDX</a> </li></ul>

a 1.5 hour procedure for identification of enterococcus species directly from blood cultures

肠球菌是一种常见的原因与大肠杆菌菌血症粪被屎肠球菌的主要品种。由于耐氨苄青霉素和万古霉素在大肠杆菌中粪仍是少见相比，屎肠球菌的阻力，快速测试，使肠球菌物种之间的分化发展是很重要的，适当的治疗和耐药性监测。的大肠杆菌粪 OE PNA FISH检测（AdvanDx，沃本，马萨诸塞州）使用物种特异性的肽核酸（PNA）探针荧光原位杂交格式，并提供了一个时间为1.5小时和特定物种提供重要信息的可能性结果治疗。多中心研究，以评估E. 1.5小时的表现粪 / OE PNA鱼程序相比原来的2.5小时的检测程序和标准的细菌学方法对肠球菌的鉴定，直接从阳性血培养瓶。

Watch this Scientific Journal Video about A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures at JoVE.com

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

一个从阳性的血液，使用一种肽核酸荧光原位杂交法（PNA FISH）文化，直接鉴定为粪肠球菌和其他肠球菌物种的快速协议。

一个1.5小时的程序，鉴定肠球菌直接从血培养的物种

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

a-15-hour-procedure-for-identification-enterococcus-species-directly

Research

JoVE Journal

Immunology and Infection

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

20.8K 次观看.  Cedars-Sinai Medical Cente. 一个从阳性的血液，使用一种肽核酸荧光原位杂交法（PNA FISH）文化，直接鉴定为粪肠球菌和其他肠球菌物种的快速协议。

视频: A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

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

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 ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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 ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

为快速细菌鉴定和药敏试验的准备血培养微丸

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...