我们感谢他的专家技术支持，哈维亨斯利博士。这项工作得益于以下的福克斯蔡斯癌症中心（FCCC）设施：实验室动物基金和小动物成像设施的使用。这项工作是支持的，一部分由“气候变化框架公约”/宾夕法尼亚大学（P50 CA083638），“气候变化框架公约”的核心授予（P30 CA006927）在卵巢癌的孢子，和卵巢癌研究基金。

一，卵巢肿瘤细胞的制备<ol><li>在文化表达荧光素酶的成长卵巢癌的细胞株。荧光素酶的来源可以是萤火虫，海肾，或其他物种。也可用于荧光蛋白的表达。对于本演示中，我们使用的卵巢癌的细胞株，OVCAR5，表达萤火虫荧光素酶。 </li><li>收获细胞，用常规的细胞培养技术。 </li><li>保持细胞悬液（10,000个细胞/μL）的磷酸盐缓冲液（PBS）冰直到注射时间。 </li></ol>二。 Intrabursal注射液这个过程需要从第二个人的援助。所有的外科手术是在无菌条件下进行。这包括穿着手术的装束和使用无菌手术器械，注射器和针头。 <ol><li>准备3毫升（100毫克/毫升）的盐酸氯胺酮，甲苯噻嗪盐酸1.6毫升（100毫克/毫升），1.5毫升（10毫克/毫升）acepromazine，20毫升0.9％氯化钠混合麻醉的解决方案。 </li><li>检查动物的识别号码和观察到的健康。麻醉与氯胺酮 - 甲苯噻嗪- acepromazine准备麻醉（IP）经腹腔注射给药量为8〜9毫升/公斤体重（BW）的动物。 </li><li>通过与动物的后爪钳或手指脚趾捏，确认该动物是在接受麻醉平原。如果有脚蹬反射，等待麻醉进行更深层次的平原，直到动物是这个程序没有响应。 </li><li>动物背侧上对着它的头和它的尾巴向你面临的无菌纱布垫。切口点位于左边或右边卵巢和中线以上。刮胡子或湿用70％酒精在切口点皮毛。 </li><li>抬起湿润的皮肤使用镊子，做一个小切口用剪刀在背内侧的位置，并直接卵巢脂肪垫以上。卵巢脂肪垫下方的腹腔壁的表面应该是可见的。脂肪垫很容易识别的白色在它周围的暗粉色组织。 </li><li>轻轻抬起腹腔壁衬，如上所述，做一个小切口（5）。 </li><li>中线切口旁放置一个无菌浸泡盐水纱布垫。找到卵巢脂肪垫，轻轻将其拉出上的纱布和休息。卵巢稳定夹紧牛头犬剪辑的脂肪垫。卵巢在解剖显微镜下，位置，让针插入输卵管小管弯曲，导致滑囊（规格30，G）的。当针插入到适当的位置，应该滑囊下可见。 </li><li>轻轻推注射器推杆注入5μL的细胞悬液之间的布尔萨和卵巢，而注射器注射部位定位。这一步需要两个人。一人推柱塞，而其他人保持针的定位。迅速取出针头穿刺部位的密封，但轻轻足够不要撕破滑囊和肾小管。滑囊应该会出现适当注射要稍微扩张。 </li><li>推出猛犬剪辑的脂肪垫，轻轻地取代生殖道进入腹腔的脂肪垫回。轻轻合上腹膜衬拉衬在较低的体壁。关闭手术订书钉或伤口剪辑皮肤。 </li><li>将复苏的动物在笼子里，并提供一个安全的热源，以避免低温和加快恢复。监测呼吸频率和易用性，肌张力的回报，并自愿移动的能力。这是走向复苏的进展的良好指标。订书钉或伤口剪辑可以去除手术后7天或以上。 </li></ol>三。口服灌胃给药<ol><li> 18〜20 G灌胃针或鼻饲管使用一个圆形的提示。灌胃针应不再比从动物的头尖的最后一根肋骨的距离。 </li><li>检查动物的识别号码和观察到的健康。轻轻地用拇指和手指的肩膀皮肤抓住颈背的动物。克制应该只是公司中脱颖而出四肢延伸到每一个侧面和出路。动物不应该能够把握针。 </li><li>动物的头，用食指轻轻拉了回来，形成了直线通过颈部和食管。支持你的拇指内的动物的回。 </li><li>轻轻的和没有力量的，插入口两侧和舌头在灌胃针。在一个平滑的运动，应通过对针口的屋顶上下食道。重力应引导针没有会议抵抗。如果有任何resistanCE，取出针，并再次启动。动物的插科打诨和吞咽反射可能会触发插入时。但是，应无阻力或动物喘气。重要的是要确保动物呼吸时，针在被检查的鼻孔和胸部的运动。 </li><li>慢慢推注射器的柱塞免除剂量体积。 </li><li>灌胃针头轻轻地取出它插入食道的方式在同样的角度和途径。 </li><li> 5〜10分钟，动物笼子里，并监测呼吸和行为。 </li></ol>四， 在活体成像我们用卡尺生命科学监察注入intrabursal腔细胞的行为。使用此系统的实验通常有4〜16周的时间内从肿瘤植入时间。 <ol><li>准备的D -荧光素（萤火虫荧光素酶的底物）的解决方案，通过溶解在1 mL PBS 5〜20毫克，并通过杀菌为0.22μm滤膜过滤。 </li><li>充电打开的氧气和异氟醚气体感应室。接通到感应室的气体流量。异氟醚的流量维持在低水平，直到动物准备进行成像。 </li><li>检查动物的识别号码和观察到的健康。放置在被控异氟醚气体感应室的动物。 </li><li>从室中取出的动物，当动物出现被麻醉。交付200μL准备通过IP使用30克针注射荧光素的解决方案。重要的是要记录注射时间，以保持一致的时间之间的荧光素注射和整个研究的成像性能。 </li><li>放置到感应室的荧光素注射的动物的回。 </li><li>成像系统设置相应的设置。要确保系统初始化之前获得的第一个动物形象。 </li><li>关掉煤气流感应室和成像室的气体流量。 </li><li>麻醉动物的背侧或腹侧了。请务必放置在鼻锥动物的鼻子，以保持适当的麻醉诱导。确保绿色照明“兴趣点网格”内的动物。 </li><li>关闭室的大门，并在适当的时候获得的图像。时间可以由动力学。 </li><li>从成像室取出的动物放到它的笼子。监视“Intrabursal注射液”中描述的动物的恢复。 </li></ol>五，代表性的成果<img src="/files/ftp_upload/2125/2125fig1.jpg" alt="图1" /> 图1。卵巢肿瘤细胞原位小鼠模型的体内成像。 intrabursally OVCAR5细胞表达荧光素酶注射到右侧卵巢，并随着时间的推移成像。图片13（左），17（中），和22（右）天后注射使用IVIS光谱成像系统。在上游面板背侧和腹侧在较低的面板。请注意，腹膜肿瘤细胞的扩散，注射后22天。 福克斯蔡斯癌症中心的机构动物护理和使用委员会规定的准则和法规的规定进行动物实验。

<ol>
	<li>Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 59, 225-249 (2009).</li><li>Lynch, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+ovarian+carcinoma:+heterogeneity,+molecular+genetics,+pathology,+and+management.">Hereditary ovarian carcinoma: heterogeneity, molecular genetics, pathology, and management.</a> Mol Oncol. 3, 97-137 (2009).</li><li>Yap, T. A., Carden, C. P., Kaye, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+chemotherapy:+targeted+therapies+in+ovarian+cancer.">Beyond chemotherapy: targeted therapies in ovarian cancer.</a> Nat Rev Cancer. 9, 167-181 (2009).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Metastasis Rev. 17, 279-284 (1998).</li><li>Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation:+advantages+and+disadvantages.">Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages.</a> Eur J Cancer. 40, 852-857 (2004).</li><li>Shaw, T. J., Senterman, M. K., Dawson, K., Crane, C. A., Vanderhyden, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intraperitoneal,+orthotopic,+and+metastatic+xenograft+models+of+human+ovarian+cancer.">Characterization of intraperitoneal, orthotopic, and metastatic xenograft models of human ovarian cancer.</a> Mol Ther. 10, 1032-1042 (2004).</li><li>Greenaway, J., Moorehead, R., Shaw, P., Petrik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-stromal+interaction+increases+cell+proliferation,+survival+and+tumorigenicity+in+a+mouse+model+of+human+epithelial+ovarian+cancer.">Epithelial-stromal interaction increases cell proliferation, survival and tumorigenicity in a mouse model of human epithelial ovarian cancer.</a> Gynecol Oncol. 108, 385-394 (2008).</li><li>Connolly, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ovarian+cancer.">Animal models of ovarian cancer.</a> Cancer Treat Res. 149, 353-391 (2009).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging.">Bioluminescence imaging.</a> Proc Am Thorac Soc. 2, 537-540 (2005).</li><li>Lehmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+and+multimodal+in+vivo+imaging+of+tumor+hypoxia+and+its+downstream+molecular+events.">Longitudinal and multimodal in vivo imaging of tumor hypoxia and its downstream molecular events.</a> Proc Natl Acad Sci U S A. 106, 14004-14009 (2009).</li><li>Connolly, D. C., Hensley, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+and+Transgenic+Mouse+Models+of+Epithelial+Ovarian+Cancer+and+Non-Invasive+Imaging+Modalities+to+Monitor+Ovarian+Tumor+Growth+In+Situ:+Applications+in+Evaluating+Novel+Therapeutic+Agents.">Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non-Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In Situ: Applications in Evaluating Novel Therapeutic Agents.</a> Current Protocols in Pharmacoogy. 45, (2009).</li></ol>

卵巢癌是所有妇科恶性肿瘤的 1，死亡的首要原因。本病的死亡率高主要是由于其已故的诊断和缺乏可靠的诊断方法 2 。此外，传统的化疗往往遇到耐药和复发癌症的3。因此，新的治疗是必需的，有效地瞄准这一疾病。针对人类卵巢癌的新疗法的发展，关键是建立一个有代表性的动物模型。 原位动物模型已经超过传统的异种移植模型的优点（如皮下或腹腔注射的肿瘤细胞），1）它再现肿瘤形成的主站点，2）它代表转移的常见部位，以及3）它提供了肿瘤细胞与适当的微环境4，5，6，7，8交互。鼠害有一个独特的法氏囊膜周围的卵巢和输卵管连续的。这种啮齿类动物的独特的解剖结构允许的卵巢肿瘤细胞的注射原位。 Intrabursally注入卵巢肿瘤细胞的行为类似于人类疾病，从而增长内intrabursal膜，并扩散到腹腔肿瘤进展。注射细胞稳定表达荧光素酶或荧光蛋白还可以实时跟踪肿瘤细胞的行为。发光法和/或荧光成像技术使人们有可能超过延长的时间和研究肿瘤的生长，分布，以及在非侵入性的方式9，10，11回归内反复形象的肿瘤细胞。 在建立原位模型，关键是迅速取出针后注入滑囊。针密封穿刺部位突然去除和防止注射细胞的泄漏。然而，在同一时间，运动应该要温柔不要撕破滑囊。如果在注射过程中发生泄漏，可能会导致腹部到种子细胞，并有可能混淆的研究，即外卵巢过早蔓延。在这种情况下，特定​​的动物应被记录下来，并为多种肿瘤部位在腹膜前处理，或者从后续分析所淘汰的发展。在执行灌胃之前，关键是检查灌胃管的长度测量，从动物的头尖最后一根肋骨。它是有帮助的，以纪念在动物的鼻管，和过去不通过这个标志管。过去这个标志的管插入可能会导致在胃的穿孔。确定管的长度是特别重要的的与年轻的动物或动物称量下20克。向下食道灌胃针插入后，应无阻力或从动物的斗争。重要的是管理的溶液或悬浮液太快，因为这可能会导致回流，并随后提供不准确的剂量体积，以及增加动物的压力。对于不时媲美的成像效果，这是最好保持注射荧光素酶的底物和成像之间的时间相一致。时间的推移，可以由基板注射后的一系列图像，观察信号的动力学。卵巢癌，口服给药，药物在体内成像原位小鼠模型的发展是必要的技术，为更好地了解卵巢癌的发展和传播，并评估新的治疗方案，最终可能会提高对病人的与这种致命的疾病结果。

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<td>Ketamine Hydrochloride</td>
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<td>Vedco, Inc.</td>
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<td>Xylazine Hydrochloride</td>
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<td>Lloyd, Inc.</td>
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<td>Acepromazine</td>
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<td>Vedco, Inc.</td>
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<td>D-Luciferin Potassium Salt</td>
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<td>Caliper Life Sciences</td>
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in vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer

更好地代表人类癌症和治疗反应的原位动物模型。本文介绍的卵巢癌的原位小鼠模型，治疗癌症的药物，通过口服给药和肿瘤细胞的行为响应实时的治疗药物在体内成像系统中使用的监测发展。在此原位模型，卵巢肿瘤细胞表达荧光素酶外用他们直接注射到老鼠囊中，其中每个卵巢封闭。萤火虫荧光素酶的底物D -荧光素，注射后，荧光素酶表达细胞产生生物发光信号。这个信号是在活体成像系统的检测和监测肿瘤的生长，分布，并在动物个体的回归的一种非侵入性的手段。通过灌胃的药物管理允许的最大加药量为10毫升/公斤体重直接传送到胃和紧密合作，类似的药物在临床治疗提供。因此，技术描述这里，卵巢癌，口服给药，药物在体内成像原位小鼠模型的发展，有助于更好地理解人类卵巢癌和治疗，并改善针对这种疾病。

Watch this Scientific Journal Video about In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer at JoVE.com

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

卵巢癌的原位动物模型复制更好的人类疾病，因此，加强我们了解癌症的发展和肿瘤对治疗的反应。一个小鼠模型接收intrabursal注射荧光素酶表达的卵巢肿瘤细胞。治疗是通过灌胃给药。监测肿瘤生长<em&gt;在体内</em&gt;成像系统。

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

in-vivo-imaging-therapeutic-treatments-an-orthotopic-mouse-model

Research

JoVE Journal

Biology

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

32.9K 次观看.  Women's Cancer Program.  中文 

视频: In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

结直肠癌原位小鼠模型

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

科研

生物学

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and future drug-eluting stents.
A variety of antibodies and transgenic and knockout strains are available in rats. Consequently, a model for in-stent restenosis in the rat would be convenient for pathobiological and pathophysiological studies. 
In this video, we present the full procedure and pit-falls of a rat stent model suitable for high throughput stent research. We will show the surgical procedure of stent deployment, and the assessment of in-stent restenosis using the most elegant technique of OCT (Optical Coherence Tomography). This technique provides high accuracy in assessing plaque CSAs (cross section areas) and correlates well with histological sections, which require special and time consuming embedding and sectioning techniques. OCT imaging further allows longitudinal monitoring of the development of in-stent restenosis within the same animal compared to one-time snapshots using histology.

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

支架内再狭窄的影像：一种廉价的，可靠，快速的临床前模型

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

3D球形模型中癌细胞入侵的时限解荧光成像和分析

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

在维沃 小鼠头骨骨髓中巨细胞和前列腺的双光子成像

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

对小鼠皮肤中的中性粒细胞迁移进行成像，以研究生理条件下的亚细胞膜重塑

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

使用 mtHyper7 对活酵母细胞中的线粒体过氧化物进行成像

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

作者聚焦：推进线粒体研究 - 用于亚细胞分析的 mtHyper7 生物传感器 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

mtHyPer7（一种用于线粒体过氧化物的比例生物传感器）在活酵母细胞中的成像

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...