Authors would like to thank Catherine Solioz for her skillful assistance.

1. BCM准备<ol><li>从获得当地的屠户尽可能新鲜的猪的下颌骨。将下颚骨到坚硬的表面，并释放一个全厚皮瓣特别注意不要留下任何软组织或骨膜附着于骨。在清洁的环境，而不需要的流罩下工作的工作。 </li><li>一旦全厚瓣被释放，使用骨刮板从颊侧收获了碎骨片。请注意，骨刮板必须锐利。处理坚决骨刮板和长运动收集骨头。弃骨碎片越小1毫米。 <ol><li>维持天然骨片，直接将骨碎片直径10厘米与补充有1％抗生素和抗真菌剂不能让它们变干的Dulbecco氏改良的Eagle氏培养基（DMEM）的塑料盘中。 </li><li>为了评价热加工的影响，受骨芯片在80℃巴氏灭菌30分钟或高压釜20分钟，121℃。 </li><li>为了评价脱矿质的影响，摇骨碎片在1M HCL为4-6小时，在4℃，并用培养基反复洗涤直到pH为中性。 </li></ol></li><li>总共5克每10ml新鲜的DMEM补充有1％抗生素和抗真菌剂的骨片的放置到一个新的塑料盘。 </li><li>将塑料皿在湿润气氛中，在37℃下放置24小时。然后，收获BCM。离心机BCM的在200×g离心10分钟以除去碎片，将其过滤灭菌（0.2纳米），并保持等份冷冻在-80℃。 </li><li>在即将使用前解冻BCM股票和避免冷冻和解冻的重复循环。 </li><li>为指示的实验中，浸泡胶原膜与BCM或无血清培养基进行1小时，在室温。用PBS洗涤大力膜并将它们放入96孔板。湿膜用细胞接种。 </li><li> BCM的制备方法概述于图1中 。 </LI&gt; </ol>基于间充质细胞生物测定2 <ol><li>种子的人类间充质细胞（例如成骨细胞，牙龈和牙周膜细胞）到12孔板用30,000个细胞/ cm 2的浓度。种子细胞用生长培养基由DMEM，10％胎牛血清和抗生素。让细胞附着到板O / N。 </li><li>弃去培养液，用预温的PBS在37℃下洗涤细胞。通过加入预热的无血清培养基中有和没有20％BCM刺激细胞。将细胞在湿润气氛中，在37℃下放置24小时。 </li><li>弃去培养液，冲洗细胞与预热PBS，并根据您的首选协议提取RNA。 </li><li>调节RNA的浓度，以使每个样品中的RNA的量相同。制备的cDNA和执行定量RT-PCR分析，使用表1所示的引物的选择的基因。 注:这是每一个组件的稀释液:2X SYBR绿，20X引着，20X引反向，5倍无菌水DD，5倍的cDNA。的定量RT-PCR在95℃15秒40个循环和60℃1分钟进行。 </li><li> CT GAPDH和Δ（ΔCT）是ΔCT刺激 - - ΔCT控制通过标准化来使用Δ（ΔCT）方法，其中ΔCT是CT目标看家基因GAPDH计算相对表达水平。 </li><li>这种质量控制后，添加BCM到培养基来刺激所有类型的细胞，包括间充质细胞，造血细胞或内皮细胞。 </li></ol>

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作者什么都没有透露。霍尔迪卡巴耶 - 塞拉诺获得奖学金的牙科研究和教育，巴塞尔，瑞士的基金会。

骨条件培养基反映骨移植物的过程中的骨再生的早期阶段的释放活性。这里描述的协议可以适于研究不同类型参与骨再生的细胞的反应。此外，该协议可以用于制备从加工骨或骨填充剂的条件培养基。该方法很容易执行，并依靠一个简单的概念:从各种天然和加工骨释放的因素。如何理解BCM影响间充质细胞可以帮助更多地了解移植整合和自体骨的性质。基于这种理念，我们已经积累了BCM从本土11,15和处理骨14,20的间充质细胞的基因表达获得的知识的影响，同时也对增殖，迁移，并进入三个主要谱系分化;成骨细胞，脂肪细胞和软骨细胞11。 BCM还检查了自己的能力，TArget造血细胞，例如相对于破骨细胞形成13的调制。许多潜在的靶细胞正在等待在体外响应BCM，这里提出的协议，可以作为引物用于这一研究。 所提出的协议也应该动画进一步揭示如何BCM激活的间质细胞尤其TGF-β调控的基因的分子机制。例如，TGF-β受体1拮抗剂SB431542阻止对基因面板ADM的表达BCM的作用，IL-11，NOX4，PRG4，和PTX3 11,15。有趣的是，碱性磷酸酶和IL33不是由SB431542 11,15表明其他目前未知的途径是通过BCM调节逆转。另一个悬而未决的问题是什么是BCM分子负责细胞反应？ BCM包含TGF-β，但并不能解释复杂的细胞反应10,11。除了​​在体外细胞方面，总体问题仍然是:向延伸确实所反映的BCM骨移植物的释放的活性，对骨再生的体内过程产生影响？从生物测定的协议和数据应该在这个方向的研究介绍。 该协议有一定的局限性。 BCM不能完全标准化，因为捐助者和收获技术之间的差异的。此外，目前在体内酶如何影响成分或BCM的活动仍是未知。未来的研究应，例如，专注于收集技术如何影响BCM的"生物活性"。上BCM的组合物骨细胞的作用也应详细研究。 BCM包含硬化，骨细胞所12日发布的几乎全部的分子。限制，但是，提供灵感的研究的下一步骤。即使与BCM研究的临床意义仍然HYpothetical，我们的协议支持的长期的概念，骨移植物，无论是天然的或处理后，释放出的"生物活性"。如何理解BCM影响细胞在体外可以推测有助于了解自体骨的工作会在体内 。

自体骨被广泛用于桥接发生作为结果畸形，切除手术，重建手术创伤，和之前植入物放置1,2-缺陷。了解骨移植物是如何支持巩固移植的过程中，生物的原则是不理解为什么自体被认为是在重建手术的金标准唯一的关键，这也是仿生骨代用品3的改进设计。尽管如此，移植合并是更快地与自体骨相比，骨替代4,5。因此，当务之急是揭示，使自体骨以便有效地支持骨再生的分子和细胞机制。 还有被认为支持整合过程6,7自体移植三种教科书的特点。首先，自体骨骨传导是，新成立的骨生长提供指导入缺陷。其次，自体骨的成骨，这意味着它包含间充质细胞，能分化成成骨细胞8。第三，自体骨是骨诱导性的像埋葬在基质骨形态发生蛋白的生长因子可以发起软骨内或甚至膜内骨形成9的过程。还有另外一个方面:新鲜制备的骨碎片保持基于在体外观察与"骨条件培养基"10-15旁分泌功能。还骨髓细胞生成的影响应该提到16。与之类似的术语"脱钙骨基质条件培养基"已经创造于1996年，支持骨旁分泌功能的整体概念，即使是由脱盐处理17。对于我们而言，BCM可以从新鲜的猪准备下颌骨10,11。 BCM的蛋白质组学分析显示的成分复杂，包括生长事实口服补液盐和细胞外基质10的成分，也延长对整骨18,19的蛋白酶现有的知识。因此，BCM应该反映骨移植体外的各种修改的发布活动。 当间质细胞，例如来自骨片或口腔软组织中分离，暴露于BCM？ 在体外会发生什么，BCM降低了成骨和脂肪细胞分化，并引发IL11表达11的强劲增长。基因组范围的微阵列显示多个基因被差异表达的间充质细胞响应于BCM。在这些基因中有肾上腺髓质素（ADM），IL11，IL33，NADPH氧化酶4（NOX4），蛋白聚糖4（PRG4或润滑素）和五聚环蛋白3（PTX3）15。从高压灭菌骨碎片得到的BCM未能改变各基因14的表达。从骨片的巴氏杀菌经历和冻结BCM能改变基因表达14。脱钙骨基质（DBM-CM）的还条件培养基改变TGF-β调控的基因20的表达。有趣的是，用于从周围的软组织21,22屏蔽骨片胶原阻挡隔膜，吸附BCM的，负责的基因表达的变化23的那些部分。 BCM研究可以扩展到参与骨再生等骨吸收的破骨细胞和内皮细胞的其它细胞类型，仅举几例。总体而言，累积体外数据提供用于临床前研究的设计的科学依据。 本协议是两方面:首先，它显示了如何准备BCM。其次，它显示了如何基于骨髓间质细胞在体外测试其生物活性。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pig Mandibles</td>
			<td>Local bucher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Scraper</td>
			<td>Hu-Friedy</td>
			<td>PPBUSE2/36</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics &#38; Antimicotics</td>
			<td>All life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Membranes (Bio-Gide)</td>
			<td>Geistlich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Invitrogen Corporation</td>
			<td align="right">16030074</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen Corporation</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			High Pure RNA Isolation Kit</td>
			<td>Roche</td>
			<td align="right">11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcriptor First Strand cDNA Synthesis Kit</td>
			<td>Roche</td>
			<td align="right">4379012001</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Microsynth</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green (for Q-RT-PCR)</td>
			<td>Roche</td>
			<td align="right">4673484001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Roche</td>
			<td align="right">11666789001</td>
			<td></td>
		</tr>
	</tbody>
</table>

bone conditioned medium: preparation and bioassay

自体骨移植被广泛应用于口腔颌面外科，骨科，骨伤。自体骨移植不仅取代失去的骨头，它们还支持骨再生的复杂过程。自体移植的这种有利的行为归因于三个特点:骨传导性，osteogenicity，和骨诱导性。然而，还有另外一个方面:骨移植物释放分子，包括生长因子，它可以靶向参与骨再生的间充质细胞的无数。骨移植物的旁分泌性能还可以通过使用骨条件培养基（BCM）来进行体外研究。在这里，我们提出了一个协议就如何从本地猪皮质骨骨准备条件培养基和骨头进行热加工或脱矿。细胞可以直接暴露于BCM或接种于生物材料，如胶原膜，与前面的BCM湿透。我们举例说明体外 bioassaYS上的TGF-β调节基因表达的间质细胞。所提出的协议应该鼓励进一步揭示骨移植的旁分泌影响骨再生期间和重建手术的广阔领域打开了转化型研究的路径。

骨条件培养基从新鲜猪碎骨片制成。该方法的一般概述，制备BCM和用于与BCM组合的生物材料示于图1和图2分别。在BCM制备，重要的是要获得大的骨片，长动作短的运动或非常小的骨片可以影响最终BCM的质量。霉素，PTX3，IL11，IL33，NOX4和PRG4（ 图3）:BCM的质量可以通过分析BCM靶基因的基因表达的控制。 ADM和PTX3都下调至0.4倍，IL11，IL33，NOX4和PRG4可以是上调至200倍。如果口腔成纤维细胞在不显示的水平不能表达BCM的靶基因，检查细胞的健康或下颌骨的新新准备BCM。 图4显示了典型的结果从BCM靶基因在接种到胶原屏障膜成纤维细胞口语表达。口服的成纤维细胞刺激从巴氏灭菌骨碎片的条件培养基，并从脱矿质骨碎片的条件培养基的20％，显示出相似的基因表达的细胞与BCM（ 表2和表3）刺激。然而，口服成纤维细胞的基因表达的从灭菌（121℃）骨片暴露于经调节的培养基，比得上未刺激对照。 <img alt="图1" src="/files/ftp_upload/52707/52707fig1.jpg" /> 图1.用于制备新鲜的猪下颌骨骨条件培养基的过程中总结。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52707/52707fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52707/52707fig2.jpg" /> 图2.摘要根据间质细胞与BCM的生物测定的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52707/52707fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52707/52707fig3.jpg" /> 图中的成纤维细胞口腔骨条件培养基靶基因3基因的表达。用来控制BCM。基因ADM和PTX3 质量六个基因典型结果下调（A）和IL11，IL33，NOX4，PRG4被上调（B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52707/52707fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52707/52707fig4.jpg" /> 图4.骨conditione的基因表达D培养基的靶基因中接种在胶原阻挡隔膜口服成纤维细胞。用于控制BCM的质量六个基因的典型结果。基因ADM和PTX3被下调（A）和IL11，NOX4，PRG4被上调（B）。根据所使用的生物材料，生长因子的吸收可以不同。胶原膜没有吸收控制IL33的表达，因此IL33表达不在此设置调节因素。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52707/52707fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><colgroup><col span="3" /></colgroup><tbody><tr><td> 缩写 </td><td> 引着 </td><td> 反向引 </td></tr><tr><td> GAPDH </td><td> AGCCACATCGCTCAGACAC </td><td> GCCCAATACGACCAAATCC </td></tr><tr><td> ADM </td><td> GGACATGAAGGGTGCCTCTC </td><td> TGTTCATGCTCTGGCGGTAG </td></tr><tr><td> IL11 </td><td> TGCACCTGACACTTGACTGG </td><td> AGTCTTCAGCAGCAGCAGTC </td></tr><tr><td> IL33 </td><td> TCAGGTGACGGTGTTGATGG </td><td> GGAGCTCCACAGAGTGTTCC </td></tr><tr><td> NOX4 </td><td> TCTTGGCTTACCTCCGAGGA </td><td> CTCCTGGTTCTCCTGCTTGG </td></tr><tr><td> PRG4 </td><td> CGACGCCCAATGTAAGAAGT </td><td> GGTGATGTGGGATTATGCACT </td></tr><tr><td> PTX3 </td><td> TGTATGTGAATTTGGACAACGAA </td><td> CATTCCGAGTGCTCCTGAC </td></tr></tbody></table> 表1:所用的6个基因的引物序列。 <table border="0" cellpadding="0" cellspacing="0"><colgroup><col span="3" /></colgroup><tbody><tr><td> 基因 </td><td> 80°C平均±SD </td><td> 121°C平均±SD </td></tr><tr><td> ADM </td><td> 0.2±0.1 </td><td> 1.1±0.2 </td></tr><tr><td> PTX3 </td><td> 0.1±0.1 </td><td> 0.9±0.2 </td></tr><tr><td> IL11 </td><td> 20±10 </td><td> 1.5±1 </td></tr><tr><td> IL33 </td><td> 15±5 </td><td> 1.2±4 </td></tr><tr><td> NOX4 </td><td> 35±15 </td><td> 2±1 </td></tr><tr><td> PRG4 </td><td> 40±10 </td><td> 1.8±1 </td></tr></tbody></table> 表2:阿霉素，PTX3，IL11，IL33，NOX4和PRG4在从热处理后的骨碎片刺激条件培养基的20％口服成纤维细胞的典型的基因表达。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 基因 </td><td> 平均±SD </td></tr><tr><td> ADM </td><td> 0.1±0.1 </td></tr><tr><td> PTX3 </td><tD&gt; 0.1±0.1 </td></tr><tr><td> IL11 </td><td> 15±5 </td></tr><tr><td> IL33 </td><td> 20±10 </td></tr><tr><td> NOX4 </td><td> 60±15 </td></tr><tr><td> PRG4 </td><td> 50±20 </td></tr></tbody></table> 表3:阿霉素，PTX3，IL11，IL33，NOX4和PRG4与来自脱矿质骨碎片的条件培养基的20％刺激的口腔成纤维细胞的典型的基因表达 。

Watch this Scientific Journal Video about Bone Conditioned Medium: Preparation and Bioassay at JoVE.com

Bone Conditioned Medium: Preparation and Bioassay

我们在这里描述了如何准备骨条件培养基（BCM）和体外测试其活性。

骨条件培养基的制备和生物测定

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

bone-conditioned-medium-preparation-and-bioassay

Nanyang Technological University

Research

JoVE Journal

Biology

11.0K 次观看.  School of Dental Medicine, University of Bern. 我们在这里描述了如何准备骨条件培养基（BCM）和体外测试其活性。 

视频: Bone Conditioned Medium: Preparation and Bioassay

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

维持细胞培养基的Wolbachia

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

组织特异性的伤害性和炎症介质的测量人体内生物活性测定

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

脱钙骨组织学，形态计量学和荧光染料分析制备

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Cryopreserving和恢复人类iPS细胞血清替代馈线中使用完整的基因敲除

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

细胞调理介质细胞外囊泡的流式细胞学分析

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

从人骨关节炎膝关节收集和RNA提取

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

使用实时细胞代谢通量分析仪监测成骨细胞生物能量学

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...