研究是由哈利·温斯顿研究基金会的支持。

1。准备新鲜的工作方案<ol><li> TBS电视台：0.05 mol的Tris缓冲液，pH值7.8公司（TBS）。准备60毫升TBS /微孔板。 </li><li> 个人防护装备：猪胰弹性蛋白酶，1型（PPE）的暂停。摇匀，稀释约0.01 U / ml的个人防护装备在TBS。准备6毫升工作液/微孔板）。 </li><li> SA的3 NA：琥珀- L-丙氨酸-L-丙氨酸- L-丙氨酸-P-硝基苯胺（SA的3，NA）。准备在二甲基亚砜（DMSO）0.1 M的股票SA 3适用的解决方案，并立即使用或储存于-20°C准备6毫升工作方案/微孔板稀释股票SA 3适用的解决方案在TBS的1/100的。 </li><li> 考马斯蓝：准备一个股票的10％考马斯亮蓝R-250（考马斯蓝）在TBS的解决方案，并使用立即或贮存于-20°C准备6毫升工作的解决方案，通过在TBS稀释原液1/1000。 </li></ol> 2。准备唾液<oL&gt; <li> 唾液收集：在站立姿势，刺激唾液置于柠檬酸或口嚼口香糖，如sialogogue血清渗出。一个无糖柠檬下降是方便的HIV-1患者频繁缺牙，无法嚼口香糖。燕子为首次3分钟需要，并收集到了接下来的3分钟的雪山杯全口唾沫。这将提供约2毫升的唾液。立即使用或储存于-80°C。 </li><li> 唾液处理：如果冷冻，在37℃，置于冰上解冻标本。新鲜或解冻标本存放在2-4°C间不超过3天。做不涡唾液和避免吸取其中包含无血清渗出的粘液。使用标准处理唾液<a href="http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf" target="_blank">http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf</a>时的注意事项。 </li></ol> 3。微孔板设置<ol><li><strONG&gt;缓冲区：一式三份进行测量。被添加到每个微孔板以及130μL，TBS电视台。 </li><li> 标本：每个测试，加入终体积为150μL/孔20μL唾液。没有引入气泡移液混合。 </li><li> 控制：阳性对照包含个人防护装备，但没有唾液。阴性对照组中含有唾液，但没有PPE.To阳性对照，以及添加20μL的TBS电视台。到负的控制，以及添加20μL的唾液。 </li><li> PPE的孵育：摇个人防护装备工作的解决方案。加入50μL的个人防护装备工作的解决方案，以每口井不包括阴性对照井。阴性对照井，添加50μL的TBS电视台。在23°C孵育10分钟</li><li> 底物孵育：3 SA的50μL，无工作的解决方案，以每口井。在23°C孵育45分钟</li><li> 考马斯蓝：50μL考马斯蓝工作液添加&lt;/ LI&gt; <li> READ-酶标仪读取吸光度，405纳米检测SA的3无乳沟代表积极α1 PI和一个595 nm的检测考马斯亮蓝蛋白质含量。 </li></ol> 4。计算α1的PI指数<ol><li> 正常化的平均405 nm 和 595 nm处 ：对于每个一式三份，计算平均值。从样本平均减去负控制平均水平。为了最大限度地减少板对板的变化，规范的平均值除以平均每个标本阳性对照平均如下： </li></ol><img alt="式（1）" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> 计算α1有价证券INEX：除以归归一个595纳米405纳米 ： </li></ol> 5。代表结果以确定是否α测试可能适合使用作为保健点的筛选试验，监测CD4细胞计数在地方病，资源有限的地区，刺激唾液收集了20位女性和11位男性的HIV-1受试者参加诊所在喀麦隆的日常护理。与α测试使用唾液收集在纽约市的发展过程中的初步意见是一致的，在喀麦隆与CD4细胞计数（R 2 = 0.91，P &lt;0.0001，N = 31）相关的α1 PI指数在唾液收集。关系的定义是由3个参数的S形曲线，具有剂量-反应关系（图2A）是一致的。 95％的信心和预测区间描绘蓝色和红色的线，分别为（图2B）。 在先前的研究中，我们确定的CD4 + T细胞表现出正弦循环周期23±3.5天在HIV-1科目，并在继承的版本与α1有价证券投资不足（PIZZ）3科目。此处所描述的是一个计算机生成的正弦曲线分析骑自行车在非HIV-1健康展出27天的周期（图3A）控制的CD4代表性的例子。 计算机生成的正弦曲线分析的CD4 + T细胞的变化，在6个科目取得了峰-峰幅度和振荡3轴的值。正如所预料的，振荡轴与振幅（R 2 = 0.96，P = 0.009，N = 6）（ 图3B）。在患者低的CD4 T细胞计数，CD4 + T细胞计数的周期性变化较小，在高CD4 + T细胞计数的患者，周期性变化大。 因为在图2所示的回归线估计振荡和振荡轴的轴与振幅，振幅可以计算的回归，从而估计多远的上方和下方的回归线CD4 + T细胞，将有望因自行车（ 图3C）。叠加图3c和图2B计算置信区间的幅度计算，人们发现，在于95％的幅度（绿线）内的置信区间（蓝线）（ 图3D）。 <img alt="图1" src="/files/ftp_upload/3999/3999fig1.jpg" /> 图1。程序简介：口广场柠檬酸和收集唾液。新增摊薄唾液微孔板一式三份的水井。新增个人防护装备，以每口井，并在23°C孵育15分钟添加PPE基板每口井，并在23°C孵育45分钟。加入考马斯亮蓝和读取在405 nm检测个人防护装备的活动，并在595 nm处检测蛋白。 <img alt="图2" src="/files/ftp_upload/3999/3999fig2.jpg" /> 图2。 α测试比较标准的CD4枚举法。一）α测试是由定量刺激的全口唾液中的α1 PI指数。医疗使用标准的流式细胞仪由一个独立的实验室进行CD4细胞计数。流式细胞仪和唾液，血液被收集在同一诊。由三参数S形曲线（R 2 = 0.91，P &lt;0.0001，N = 31），B）95％的信心和预测区间在蓝色和红色描绘的α1 PI指数和CD4细胞计数之间的关系被定义线，分别。 <img alt="图3" src="/files/ftp_upload/3999/3999fig3.jpg" /> 图3。精度计算的CD4Ç使用的α1PIIndex.A的ounts）的CD4 + T细胞具有非HIV-1健康控制在27天周期的正弦循环。描绘振荡轴（红色虚线）B）计算机生成的CD4 T细胞循环4 HIV-1的科目是用来计算的峰-峰值的幅度和振荡轴，非HIV-1的正弦波分析-1 PIzz主体，非HIV-1健康对照A部分描述的CD4之间的关系的T +细胞的幅度和振荡轴的定义是由3个参数的S形曲线（R 2 = 0.96，P = 0.009，N = 6），C）B部分）的振荡幅度和轴之间的S形关系被用来计算预期的振幅沿回归线（黑线）从图2点（绿线）D）预期幅度间隔（绿线）从95％置信区间显着差异（蓝线）曼惠特尼秩和检验（中位数95％的置信区间和回归线（63细胞/μL之间的差异比较基础）之间的振幅线，回归线（37细胞中位数差异决定/μL），P = 0.001）。

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没有利益冲突的声明。

未稀释的血清中含有的中位数为36μM的α1 PI和3.6微米α2巨球蛋白（α2米 ），10倍的差异8。这两种蛋白质的竞争结合个人防护装备。利用这两个特点，浓度和个人防护装备的亲和力，已发展出一种方法，是专门测量在正常血清中存在竞争α2米 8的α1有价证券。这种方法可以在0.3％血清检测低至50纳米μ1有价证券。在血清稀释度为2.5...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> sialogogue，0.05M柠檬酸</td><td> EDA的无糖糖果，4900后方N.20th街，费城，PA的19144 </td><td>无糖柠檬滴</td></tr><tr><td>猪胰弹性蛋白酶，1型（欧共体3.4.21.36） </td><td> Sigma-Aldrich公司，圣路易斯，密苏里州63103 </td><td> E1250 </td></tr><tr><td>琥珀 - L-丙氨酸-L-丙氨酸 - L-丙氨酸-P-硝基苯胺</td><td> Sigma-Aldrich公司</td><td> S4760 </td></tr><tr><td>考马斯亮蓝R-250 </td><td> Sigma-Aldrich公司</td><td> B7920 </td></tr><tr><td>组织培养处理，96孔板</td><td>碧迪，富兰克林湖，NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td>酶标仪405 nm和595 nm的过滤器</td><td> MTX的实验室系统，8456泰科路，裴lding研发，维也纳，弗吉尼亚州22182 </td><td> Dynex的P97277 </td></tr></tbody></table>

the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva

有负担得起的CD4枚举监测HIV疾病的迫切需要。的CD4枚举是在资源有限的地区，由于时间和温度的限制，技术复杂，试剂成本，尤其是单克隆抗体的血细胞，目前唯一可以接受的方法来衡量的CD4遥不可及。节约成本和节省时间的一种常用实验室的策略，而不是衡量某些血液值，是计算。例如，低密度脂蛋白水平计算，总胆固醇，高密度脂蛋白（HDL），甘油三酯1使用的测量水平。因此，确定无细胞的相关因素，直接调节的CD4 + T细胞可以提供一个准确的方法，计算CD4计数由于生理意义的相关因素。 进入血液，并注定要成为循环CD4 + T细胞的干细胞数量确定的趋化因子CXCL12的1ND及其受体CXCR4，由于他们对运动2的影响。此外，进入血液干细胞的运动过程调节细胞表面人类白细胞弹性蛋白酶（HLE CS）和HLE CS-反应活性α1蛋白酶抑制剂（α1有价证券，α1抗胰蛋白酶，SerpinA1）3。在HIV-1病毒病，α1 PI灭活由于疾病的进程4。 α1 PI在HIV-1疾病的早期症状类别，积极发现低于100％未经治疗的HIV-1患者（中位数= 12μM，以及达到正常水平，在对症4大类，5正常。格局已被归因于免疫灭活，不足够的合成，蛋白失活，或充氧。我们观察到，在HIV-1&gt; 220的CD4细胞/μl的科目，CD4细胞计数了积极的α1 PI水平呈正相关（R 2 = 0.93，P＆LT; 0.0001，N = 26）和无效的α1的PI（R 2 = 0.91，P &lt;0.0001，N = 26）5。 α1的PI HIV-1感染和未感染者的管理，导致在α1 PI参与调节的CD4 + T细胞，血液中的3中的CD4计数显着增加。 与刺激，全唾液中含有足够的浆液性渗出物（血浆含有蛋白质的物质通过血管壁进入唾液传递）允许测量活性α1 PI与此测量的相关性的证据表明，它是一种精确的方法计算CD4细胞计数。简言之，如嚼口香糖或柠檬酸sialogogues刺激到整个口腔唾液，血清渗出。刺激血清渗出后，血清α1 PI唾液中的活性是衡量其能力，抑制弹性蛋白酶的活性。猪胰弹性蛋白酶（PPE）的弹性蛋白酶是一个现成的廉价来源。个人防护装备的α1有价证券结合形成一个到一个复杂的，防止切割其特定的底物，其中之一是的比色法肽，琥珀- L-丙氨酸-L-丙氨酸- L-丙氨酸-P-硝基苯胺（SA的个人防护装备3不适用）。孵化器为在室温下10分钟的个人防护装备的饱和浓度的唾液，允许个人防护装备的所有活动α1 PI唾液中的约束力。可以衡量的PPE抑制活性α1有价证券加入的PPE基板SA的3不适用。（图1）。虽然CD4细胞计数测量血容量（CD4细胞/μL），α1 PI唾液中的浓度与唾液中的血清浓度，唾液量，因为体积可以改变白天和人大大6人。然而，几乎所有的唾液中的蛋白质是由于血清含量，唾液中的蛋白质含量是测量surable 7。因此，活性α1 PI唾液中的唾液蛋白质含量的比率计算，被称为α1 PI指数。结果提出此表明，α1 PI指数提供了一个准确和精确的计算CD4细胞计数的生理方法。

Watch this Scientific Journal Video about The Î±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

被描述的CD4枚举法，α测试，它采用全唾液，以提供快速，准确的CD4细胞计数。 α-测试成本便士和消除技术培训，如昂贵的试剂，单克隆抗体，仪器仪表，制冷，样品运输，以及收集和处理血液的需要。

测试：α-细胞快速免费的CD4计数，使用全唾液

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

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The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

13.7K 次观看.  Weill Cornell Medical College. 被描述的CD4枚举法，α测试，它采用全唾液，以提供快速，准确的CD4细胞计数。 α-测试成本便士和消除技术培训，如昂贵的试剂，单克隆抗体，仪器仪表，制冷，样品运输，以及收集和处理血液的需要。

视频: The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

科研

医学

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

快速点护理依诺肝素的抗凝血剂功效的测定全血中

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

采样使用超探头棒棒糖样的土著人的唾液多肽：简化和增强肽的临床质谱检测

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

3D 细胞培养和非抗凝全血对内皮细胞抗凝及抗炎作用的评价

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

酸化浸渍法评价全肾肾素数

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is &#60;10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

使用台式流变仪快速表征气道粘液的粘弹性

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

Whole-Kidney Three-Dimensional Staining with CUBIC

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.