我们感谢美国国家癌症研究所IMAT计划从美国国立卫生研究院对这项工作[R33 CA174616-01A1]资金。

1.目标细胞的制备<ol><li>在37℃下保持的靶细胞（ 例如 ，对Raji淋巴瘤细胞）的生长培养基（RPMI 1640培养基，含10％热灭活的胎牛血清（FBS），4nm的L-谷氨酰胺，和500单位/ ml青霉素/链霉素）的在一个5％CO 2的培养箱中培养。 </li><li>离心样品以沉淀靶细胞在15ml试管中。离心时间随不同的细胞类型;离心3分钟，在468×g离心Raji细胞。 </li><li>第一次吸上形成的上清液表面有气泡，并删除整个上清。 </li><li>加入10毫升的PBS到管。通过吸取的溶液上下数次重新悬浮细胞，以打散细胞丛。 </li><li>离心机的样品为3分钟，在468×g下。 </li><li>吸出形成的上层清液的表面上的任何气泡，并删除整个上清液。 </li></ol> 2.制备细胞微阵列<ol><li> Øbtain细胞附着96孔板中包或单一单元阵列8孔腔室玻片（见表材料/试剂）。 </li><li>适用于1.2微升活化剂从工具包中的DNA试剂。盖小瓶，反其道而行，并轻轻摇动使溶液混合。 </li><li>使该溶液20分钟，在室温下进行反应。 </li><li>适用于该混合溶液中的靶细胞沉淀（在15ml试管中）。 </li><li>添加绿色荧光胞浆染料以500nM的最终浓度。 </li><li>孵育靶细胞与在定轨振荡器的混合溶液和染料为在室温30分钟。 </li><li>用5毫升的PBS洗涤细胞两次，并将细胞重新悬浮在10ml的生长培养基。 </li><li>从步骤2.7向每孔中应用100μl的溶液。用血细胞计数器计数细胞数。确保细胞数，每孔介于5×10 4〜1×10 6个细胞。 </li><li>后在室温下孵育10-15分钟，轻轻两次200-30洗样品0微升生长培养基以除去任何未结合的细胞。 注：关于5-10微升生长培养基洗涤后残留在各孔中。 </li></ol> 3.全血细胞毒性分析<ol><li>加入180微升的人全血，以每个样品井。 </li><li>获得抗CD20抗体溶液，在5毫克/毫升，并执行连续稀释，使50，20，10，5,2，1，在1.5ml管中抗CD20抗体溶液0.5微克/毫升。加入20μl抗CD20抗体的各个浓度的每个样品井制备一式三份。 </li><li>加入碘化丙啶以500nM的最终浓度，以每孔中。 </li><li>培养所得到的板在37℃，5％CO 2的16小时。 </li><li>图像上的幻灯片或96孔板，支持8倍的放大倍率自动成像系统的细胞。 </li><li>使用自动成像系统的细胞计数程序进行量化的结果。 注：自动成像系统的软件提供简单一步一步进行细胞计数操作。使用该软件的协议可以在供应商的网站上找到。 </li><li>从该软件的主界面[查看结果。 </li><li>选择[保存的样本文件。 </li><li>第一门FITC平均强度&gt; 50，则门TxRed平均强度&gt; 20。 </li><li>选择[板块综述]。 </li><li>选择[导出表。 注：该方案的百分比的细胞杀伤式如下所示： ％靶细胞杀伤=（＃细胞染色的绿色和红色的/＃细胞染成绿色）* 100％ </li></ol>

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作者有没有竞争的财务权益。

WCA的体外抗肿瘤筛查工具与单细胞分辨率12-16，传统的放映目标，如CDC和ADCC分析9-11，和之前的临床前动物试验后，最好利用的关键。目前，主要的靶标筛选测定，如CDC或ADCC检测在一个简化的介质或缓冲体系都执行。然而，候选药物，显示功效在这些简化的缓冲系统并不总是有效的，在更复杂的全血系统。因此，WCA可以桥接传统的目标掩护和昂贵的动物研究之间的间隙，减...

在制药工业中的最新进展已经导致了在实现肿瘤细胞抗体和个性化的癌症治疗方法的具体标识的兴趣增加;然而，几个障碍物的过程中遇到。只有5％是在临床前开发的抗癌活性剂都出现在阶段II-III测试1,2足够的疗效后，获得许可。临床前的策略（ 在体外和体内 ）是次优的作为许多例子已经表明，抗肿瘤药物有不同的行为在人类和实验动物的主要是由于其不同的血液成分3-5。 为解决抗肿瘤药物筛选平台的必要性，并提供昂贵的动物实验和临床试验前一个检查点，一个人全血细胞毒性测定法（WCA），提出了用于评价抗肿瘤药物疗效以更加相关的生物环境。该全血中的细胞毒性测定法可用于评估个体的细胞与抗体和其他药物候选物在人全血的响应。 的WCA是通过将高通量细胞定位技术与高通量和高含量成像6开发的。通过利用一种自动成像系统中，两个活细胞和死细胞的数目可以用较高的精确度来确定。由于这一事实，即靶细胞被固定在同一焦平面上，WCA是能够提供实时定量细胞毒性分析，而无需去除红细胞。此外，该自动成像系统提供了若干优点，如已达到指定的搜索条件的唯一的靶细胞（ 例如 ，荧光标记的细胞和细胞形态）被选通，并进行处理。此外，它允许生产每天144的板。因此，这种成像能力和吞吐量允许高-C的运行ontent和同时高通量实验。通过组合的WCA和自动成像系统，高通量定量细胞的细胞毒性分析可以在一个以上生物相关的环境来实现。

<table border="0" cellpadding="0" cellspacing="0" width="761">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Name of Material/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:135px;">Name/Discription</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Cell attachment 96 well plate kits&#160;</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">AP9601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Suspension Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0801</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Adherent Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20+</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CCL-86</td>
			<td style="width:135px;">Raji cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20-</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL-8083</td>
			<td style="width:135px;">MC/CAR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">RPMI 1640 with L-glutamine</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11875-119</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Thermo</td>
			<td style="width:119px;">SH30070.01HI</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Peni/Strep</td>
			<td style="width:172px;">Life Technologies</td>
			<td align="right" style="width:119px;">15070063</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cytosolic dye</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">C7025</td>
			<td style="width:135px;">Cell Tracker Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Rituxan (Biosimilar)&#160;</td>
			<td style="width:172px;">Eureka Therapeutics</td>
			<td style="width:119px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Human whole blood</td>
			<td style="width:172px;">Allcells</td>
			<td style="width:119px;">WB001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Propidium Iodide</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:119px;">P4170-10MG</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Automatic imaging system</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
			<td style="width:135px;">Cell Reporter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cell counting program</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
			<td style="width:135px;">Cell Reporter</td>
		</tr>
	</tbody>
</table>

real-time cytotoxicity assays in human whole blood

的实时的基于细胞的全血中的细胞毒性测定法（WCA），允许访问整个细胞的细胞毒作用的时间信息被开发的高通量细胞定位技术。靶向肿瘤细胞群体的第一预编程，以固定到一个数组的格式，并标记有绿色荧光胞浆染料。以下的单元阵列形成，抗体药物的加入与人全血组合。碘化丙啶（PI），然后加入以评估细胞死亡。单元阵列是用自动成像系统进行分析。而细胞质染料标记的靶向肿瘤细胞群，PI标签死肿瘤细胞群。因此，靶癌细胞的细胞杀伤百分比可通过计算存活的靶细胞，以死靶向细胞的数目的数量进行定量。用这种方法，研究人员能够访问时间依赖性和剂量依赖性细胞的细胞毒性的信息。值得注意的是，没有任何有害的无线电化学品的使用。这里介绍的WCA已经过测试与淋巴瘤，白血病和实体瘤细胞系。因此，WCA可以让研究人员评估在一个高度相关的离体条件下的药物的疗效。

抗CD20抗体和淋巴瘤细胞（Raji细胞和MC / CAR）被选择作为模型系统来证明全血细胞毒性测定法（WCA）7,8。 Raji细胞有CD20的高拷贝数的在细胞表面上，而MC / CAR的细胞有CD20的低拷贝数的对它们的膜。靶向细胞首先染成绿色与绿色荧光细胞内的染料，并排列在96孔板中。万 - 50000靶淋巴瘤细胞被固定在每个孔中。 180微升新鲜抽取的人全血加入到每孔中。抗CD20抗体，然后加入到96孔板中以梯度浓?...

Watch this Scientific Journal Video about Real-time Cytotoxicity Assays in Human Whole Blood at JoVE.com

Real-time Cytotoxicity Assays in Human Whole Blood

全血细胞毒性测定（WCA）是通过将高通量细胞定位技术与荧光显微镜和自动化图像处理开发了细胞毒性测定。在这里，我们描述了如何用抗CD20抗体治疗的淋巴瘤细胞可以分析实时人全血中，以提供定量细胞的细胞毒性分析。

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 ...

real-time-cytotoxicity-assays-in-human-whole-blood

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12.1K 次观看.  Adheren, Inc. 全血细胞毒性测定（WCA）是通过将高通量细胞定位技术与荧光显微镜和自动化图像处理开发了细胞毒性测定。在这里，我们描述了如何用抗CD20抗体治疗的淋巴瘤细胞可以分析实时人全血中，以提供定量细胞的细胞毒性分析。 

视频: Real-time Cytotoxicity Assays in Human Whole Blood

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

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 ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

的运动再培训采用实时反馈的性能

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

在新生小鼠肺肺流体体积的实时X射线成像

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

通过实时血液动力学监测在小鼠中通过肝脏浸润建立的不受控制的出血性休克

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...