这项研究是支持的关节炎和肌肉骨骼及皮肤疾病，美国国立卫生研究院国立研究所院内研究计划。

1。收集全血<ol><li>获取从受影响的个人，以及年龄和性别匹配的健康对照最小的10毫升外周血。静脉血应收集到，而不是使用其他抗凝血剂，如EDTA，倒几次，以确保适当的混合与肝素钠肝素钠真空采血管的血液收集管。请注意，10毫升是体积最小的电镀样品需要重复这个特殊的检测全血。 </li><li>样品应尽快处理收集后，以取得最佳效果。但是，肝素血液可以存储在昏暗的灯光下下，长达24小时，直到准备好要处理的（2）在室温下。 </li></ol> 2。全血制备<ol><li>离心机在室温下10分钟3000转（带刹车关闭）的所有血液样本。互不干扰的棕黄色大衣，等离子可以从上面收集和储存其他实验目的。血浆样品可以暂时储存在-20 ° C或-80 ° C下长期贮存</li><li>一旦血浆收集，洗样品resuspending无血清（不全）的RPMI 1640介质（环境温度），红细胞（RBC）的颗粒和巴菲大衣。轻轻地在收集管吸液管的混合物，并转移至50 mL锥形管。 </li><li>音量高达50毫升不完整的RPMI 1640培养基，轻轻颠倒管创建一个均匀混合物。离心10分钟3000转（带刹车关闭）的样品。确保有清晰的分离，吸出上清液，小心不要去打扰捉鬼外套。重复这个过程2-4次，以确保所有污染物和约束因子刺激前删除。 </li><li>最后一次洗涤后，悬浮红细胞颗粒和棕黄色的外衣，使总体积为20毫升（10毫升最初制定的全血）与不完整的RPMI 1640媒体。基于最初制定的血液量，总量可以调整。 </li><li>使用Cellometer的愿景，可稀释血液无裂解红细胞计数，通过选择使用吖啶橙（AO）的白细胞计数选项。添加一个部分稀释血液的一部分AO和负载20μL1:1混合成一个一次性的计数室。 AO的终浓度稀释后，应1μg/mL。 </li><li>根据实际情况调整不完整的RPMI 1640媒体到最终细胞浓度为2.0 × 10 6细胞/ mL。 </li></ol> 3。全血的刺激<ol><li>检测需要至少四个条件（每个重复做）：未刺激，除了单独的LPS，ATP的单独此外，终于LPS加ATP的加在一起。添加1毫升准备好的2.0 × 10 6细胞/ ml稀释全血到24孔组织培养板的适当以及。 </li><li>兴奋剂应改组，并提出了到室温开始之前的刺激实验。冻干毒素和ATP应根据制造商的指示改组。经过重组，LPS和ATP可进一步稀释到一个适当的股权集中使用不完整的RPMI 1640媒体的。 </li><li>加入脂多糖LPS的仅以及脂多糖加ATP使LPS浓度的最后工作是1微克/毫升。刺激的总时间为3小时。然而，全血是刺激与LPS 2小时40分钟。样品应放置在孵化器在37 ° C和5％的CO 2。 </li><li> ATP过程中添加3小时刺激期剩下的20分钟。 ATP ATP单独LPS和ATP良好，在孵化器的地方样本。最后工作ATP浓度为2mm。 </li><li> 3个小时的潜伏期后，收集上清aliquoting每个样品1.5 ml离心管，随后离心样品在室温下2分钟的转速在10000。上清转移到另一1.5离心管。 </li><li>样品可以立即检测或存储暂时在-20 ° C或-80 ° C下长期贮存。 </li></ol> 4。细胞因子含量<ol><li>被冻结的样品需要分析前平衡至室温。细胞因子浓度的IL -1βcan使用多种方法进行评估。我们利用生物复杂的Pro X - plex的人类细胞因子与生物复杂的200系统的检测，每制造商的说明。在这个协议中，我们是专门测量的IL -1β，但是，我们利用这种多重细胞因子的检测，为了同时测量多种细胞因子，使用一个小样本量的好处之一检测。 </li></ol> 5。代表性的成果 LPS和ATP成功的刺激后IL -1β生产的分析表明，一做SE的反应依赖于受到刺激的细胞数量。我们发现，2.0 × 10 6细胞/毫升是一个足够的浓度，以产生最佳的IL -1β和可衡量的的浓度以及其他细胞因子（图2 ）。 全血分析研究目前autoinflammatory表现的患者，是有用的。这些患者可能会出现在先天免疫系统，生产过剩（图3）与LPS单独刺激（与对照组相比）的IL -1β后，可以通过特征的缺陷。 <img src="/files/ftp_upload/2662/2662fig1.jpg" alt="图1" /> 图1。AO染色核白血细胞，以用来确定细胞浓度稀释的全血标本的形象代表。 <img src="/files/ftp_upload/2662/2662fig2.jpg" alt="图2" /> 图2，IL -1β生产从一个健康捐赠者增加细胞浓度的脂多糖刺激后测定全血上清。被检测的样品一式三份。 <img src="/files/ftp_upload/2662/2662fig3.jpg" alt="图3" /> 图3，两个健康的正常对照组，以及两个患者的IL -1β相关autoinflammatory表现呈现在全血上清测IL -1β分泌的代表结果。

<ol>
	<li>Martinon, F., Mayor, A., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasomes:+guardians+of+the+body.">The inflammasomes: guardians of the body.</a> Annu Rev Immunol. 27, 229-265 (2009).</li><li>Thurm, C. W., Halsey, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cytokine+production+using+whole+blood.">Measurement of cytokine production using whole blood.</a> Curr Protoc Immunol. 66, 7-18 .</li><li>Mariathasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopyrin+activates+the+inflammasome+in+response+to+toxins+and+ATP.">Cryopyrin activates the inflammasome in response to toxins and ATP.</a> Nature. 440, 228-232 (2006).</li><li>Gabay, C., Lamacchia, C., Palmer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1+pathways+in+inflammation+and+human+diseases.">IL-1 pathways in inflammation and human diseases.</a> Nat Rev Rheumatol. 4, 232-241 (2010).</li><li>Franchi, L., Elgenbrod, T., Mu&#241;oz-Planillo, R., Nu&#241;ez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat Immunol. 3, 241-247 (2009).</li><li>Sims, J. E., Smith, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL-1+family:+regulators+of+immunity.">The IL-1 family: regulators of immunity.</a> Nat Rev Immunol. 2, 89-102 (2010).</li><li>Haug, R., Jo&#248;, G. B., Westvik, A. B., Kierulf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+production+and+pattern+recognition+receptor+(PRR)+expression+in+whole+blood+stimulated+with+pathogen-associated+molecular+patterns+(PAMPs.">Chemokine production and pattern recognition receptor (PRR) expression in whole blood stimulated with pathogen-associated molecular patterns (PAMPs.</a> Cytokine. 32, 304-315 (2005).</li><li>Netea, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+requirement+for+the+activation+of+the+inflammasome+for+processing+and+release+of+IL-1bin+monocytes+and+macrophages.">Differential requirement for the activation of the inflammasome for processing and release of IL-1bin monocytes and macrophages.</a> Blood. 113, 2324-2335 (2009).</li><li>Latz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasomes:+mechanisms+of+activation+and+function.">The inflammasomes: mechanisms of activation and function.</a> Curr Opin Immnol. 22, 28-33 (2010).</li><li>Masters, S. L., Simon, A., Aksentijevich, I., Kastner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horror+autoinflammaticus:+the+molecular+pathophysiology+of+autoinflammtory+disease.">Horror autoinflammaticus: the molecular pathophysiology of autoinflammtory disease.</a> Annu Rev Immunol. 27, 621-668 (2009).</li></ol>

整个血液检测是一个简单的方法，更加紧密地与其他的技术，将要求细胞的分离和广泛的来样加工和操纵相比，模仿生理条件。这种方法利用全血中的白细胞计数，因此，提供了一种基于细胞的数量要准确规范样品与AO Cellometer视觉。 必须指出，这个实验中，全血应卷入肝素钠收集管其他抗凝血剂已知钙螯合剂，从而影响检测结果。血清自由的媒体也应该使用，因为这可能会?...

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		<th>Catalogue Number</th>
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<tr>
<td>Sodium Heparin Blood Collection Tubes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>366480</td>
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<td>&nbsp;</td>
<td>GIBCO</td>
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<td>Cellometer Vision</td>
<td>&nbsp;</td>
<td>Nexcelom Bioscience</td>
<td>&nbsp;</td>
<td>Contact company for instrument information</td>
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<td>Cellometer Cell Counting Chambers</td>
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<td>Nexcelom Bioscience</td>
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<td>24-well Tissue Culture Plate</td>
<td>&nbsp;</td>
<td>Corning Costar</td>
<td>3524</td>
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<td>Ultra Pure E. coli K12 LPS</td>
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<td>Adenosine 5&#180;-tripohasphate disodium salt hydrate</td>
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<td>Bio-plex 200 system</td>
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<td>Contact company for instrument information</td>
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<td>Bio-Plex Pro human cytokine x-plex assay</td>
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<td>Bio-Rad</td>
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accurate and simple measurement of the pro-inflammatory cytokine il-1&beta; using a whole blood stimulation assay

从免疫细胞可溶性介质的分泌导致的炎症过程，导致各种表现在皮肤，关节和其他组织以及改变细胞因子的动态平衡。先天免疫系统中起着至关重要的作用在识别病原体和其他内源性危险刺激。先天免疫细胞释放的主要因子之一，是白细胞介素（IL）-1。因此，我们利用全血刺激试验，以衡量炎性细胞因子的分泌和专门的促炎症细胞因子IL -1β1，2，3 。 与先天免疫系统，导致autoinflammatory综合征的遗传功能障碍的患者表现夸张成熟后，单纯用LPS刺激IL -1β的释放。为了评估先天免疫与炎症相关的病症的患者组成部分，我们使用一个特定的免疫检测病原体相关分子模式（PAMPS），如革兰阴性细菌内毒素，细胞免疫反应，脂多糖（LPS ）。这些PAMPS上的先天免疫系统4，5，6，7细胞中发现的病原体识别受体（PRRS），确认。原始信号，脂多糖，结合次要的信号，ATP，是必要的炎性激活，一个多蛋白复合物，处理亲IL -1β，其成熟， 生物活性，4，5，6，8 ，9， 10。 全血检测需要最少的样品操作，以评估需要劳力密集的隔离和特定的细胞群的培养等方法相比，细胞因子的产生。此方法不同于其他刺激全血化验的RPMI媒体的比例与稀释的样品，而不是我们执行一个白细胞计数直接从稀释的全血，因此，刺激的白血细胞在培养2。这个特殊的全血检测结果表明了一种新技术，有助于阐明病人同伙提出autoinflammatory pathophysiologies。

Watch this Scientific Journal Video about Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1Î² using a Whole Blood Stimulation Assay at JoVE.com

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

我们描述了一个简单的免疫autoinflammatory表型的患者，来衡量生产促炎性细胞因子，如IL -1β生产。通过激活细胞与病原体相关分子模式，特别是与脂多糖，全血培养，细胞因子的分泌，可以方便地评估全血上清。

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 ...

accurate-simple-measurement-pro-inflammatory-cytokine-il-1-using

Research

JoVE Journal

Medicine

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

26.2K 次观看.  National Institute of Arthritis and Musculoskeletal and Skin Diseases. 我们描述了一个简单的免疫autoinflammatory表型的患者，来衡量生产促炎性细胞因子，如IL -1β生产。通过激活细胞与病原体相关分子模式，特别是与脂多糖，全血培养，细胞因子的分泌，可以方便地评估全血上清。

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

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

在单绒毛膜胎盘血管吻合用有色染料的精确和简单的评价

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

科研

医学

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

硬膜外颅内压测量中使用的光纤压力传感器的大鼠

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

快速，准确的呼出气测量氨

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

一个简单的设备，迅速准备小鼠肠的全坐骑

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

自由活动小鼠使用遥测长期血压测量

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

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 ...