这项研究得到了韩国食品研究所（KFRI）的主要研究计划（E0210702-03）的支持，该计划由科学和信息通信技术部资助。

1.细菌接种物的制备
<ol><li>取出储存在-80°C的甘油储备瓶。</li>
	<li>使用无菌移液器吸头从小瓶中取出细菌菌株（2-10μL）。 注： 不动杆菌 菌株， 布维 曲霉（13-1-1）， 朱氏 曲霉（13-1-2）， 皮蒂 曲霉（13-2-5）， 鲍曼 曲霉（13-2-9）， 放射性曲霉 （20-1）和 乌辛曲 霉（24-1）用于本协议。</li>
	<li>用细菌接种市售的血琼脂平板（补充有5%羊血的胰蛋白酶大豆琼脂，见 材料表）。 注意：也可以使用其他培养基，例如营养琼脂或MacConkey琼脂。</li>
	<li>使用间歇性燃烧的无菌环在琼脂表面划线以使其变薄。</li>
	<li>将琼脂平板倒置在培养箱中，并在30°C下孵育20-24小时。</li>
	<li>用无菌针头挑选细菌培养物的单个菌落，并用培养物接种5mL无菌脑心脏输注液（BHI，参见 材料表）。</li>
	<li>将接种的肉汤在30°C的振荡培养箱中以约150rpm孵育20-24小时。</li>
	<li>将 1 mL 过夜培养物转移到无菌微量离心管中，并在室温 （RT） 下以 6,000 × g 离心过夜培养物 10 分钟。</li>
	<li>用移液管除去上清液。</li>
	<li>使用涡旋，将沉淀重悬于 1 mL 无菌磷酸盐缓冲盐水 （PBS） 中。</li>
	<li>在室温下以6,000× g 离心10分钟，除去上清液。</li>
	<li>在600nm处使用涡旋至约0.1的光密度，将沉淀重悬于无菌的10倍稀释BHI中。 注：约 0.1 的光密度相当于约 107 CFU/mL。</li>
</ol>2. 使用结晶紫进行生物膜定量
<ol><li>向无菌聚苯乙烯 96 孔板的每个孔中加入 200 μL 接种物（参见 材料表），并向最外层的每个孔中加入 200 μL 去离子水以防止内孔变干15.</li>
	<li>向同一板的每个孔中加入 200 μL 10 倍稀释的 BHI 作为阴性对照。</li>
	<li>将板在25°C孵育24小时。</li>
	<li>用移液管除去上清液。 注意：多通道移液器通常更方便用于多个样品。</li>
	<li>小心地向每个孔中加入300μL PBS，并用移液管除去上清液。 注意：多通道移液器通常更方便用于多个样品。</li>
	<li>再重复步骤 2.5 两次。</li>
	<li>向每个孔中加入 200 μL 结晶紫溶液 （1%）（参见 材料表），并在室温下孵育 30 分钟。</li>
	<li>重复步骤 2.4-2.6。</li>
	<li>向每个孔中加入 200 μL 无水乙醇，并在室温下孵育 15 分钟，通过上下移液数次彻底混合。</li>
	<li>将 100 μL 洗脱液从每个孔转移到新的 96 孔板中。</li>
	<li>使用酶标仪测量595nm处的吸光度（参见 材料表）。</li>
	<li>当吸光度超过2.0时，将样品在无水乙醇中适当稀释至2.0以下的吸光度，并按步骤2.11进行测量。然后，通过将观察值乘以稀释因子来计算样品的吸光度（最终值）。</li>
	<li>通过从同一孔板上测试样品的每个孔的值中减去阴性对照的平均值来计算样品的真实吸光度。</li>
</ol>3. 生物膜活力计数
<ol><li>向无菌聚苯乙烯 12 孔板15 的每个孔中加入 1 mL 接种物（步骤 1）。将板在25°C孵育24小时。用移液管除去上清液。</li>
	<li>小心地向每个孔中加入 1.5 mL 无菌 PBS，并用移液管除去上清液。向每个孔中加入 1 mL 无菌 PBS。</li>
	<li>使用安装的细胞刮刀刮掉孔的底部和壁面。</li>
	<li>将收获的细胞悬液转移到含有 9 mL 盐水 （0.85% NaCl） 和 10-20 个玻璃珠的无菌塑料管 （10-1） 中（参见 材料表）。 注意：要收集残留在孔底面上的任何残留生物膜碎片，可以将其重新悬浮并使用10-1 管中的生理盐水通过移液管转移。</li>
	<li>以最大速度涡旋10-1 管60秒。</li>
	<li>通过将 1 mL 转移到下一个含有 9 mL 盐水 （0.85 % NaCl） 至 10-7 的无菌管 （10-2） 中，进行 10 倍的连续稀释。</li>
	<li>将每种稀释液中的100μL涂布在不动杆菌琼脂上（参见 材料表），并将琼脂平板在30°C下孵育24-42小时。</li>
	<li>手动计算每个板上典型红色菌落的数量，范围在 10 到 250 之间。</li>
	<li>使用以下公式计算每个孔的生物膜中的活细胞数： 活细胞 = 菌落数 × 稀释因子 × 10</li>
</ol>4. 使用共聚焦激光扫描显微镜（CLSM）进行生物膜可视化
<ol><li>向用于显微镜分析的无菌 96 孔板的每个孔中加入 200 μL 接种物。</li>
	<li>将板在25°C孵育24小时。</li>
	<li>用移液管除去上清液。</li>
	<li>小心地向每个孔中加入 300 μL 过滤灭菌的去离子水，并用移液管除去上清液。</li>
	<li>重复步骤 4.4。</li>
	<li>制备在过滤灭菌的去离子水中稀释至最终浓度分别为10μM和60μM的SYTO 9和碘化丙啶（见 材料表）的混合物。</li>
	<li>将混合物以 200 μL 加入每个孔中，并在室温下在黑暗中孵育板 20-30 分钟。</li>
	<li>重复步骤 4.3-4.4</li>
	<li>使用激发波长分别为 488 nm 和 561 nm，发射波长范围为 499-535 nm 和 568-712 nm 的 CLSM 可视化底表面附着的生物膜。</li>
</ol>

不动杆菌生物膜形成的评估和生物膜的表征

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作者没有什么可透露的。

使用所描述的方案，测量、可视化不同程度的不动杆菌分离株的生物膜形成，并估计生物膜中的活细胞（图1、图2和图3）。
在该方案中，使用两种不同的温度，30°C用于生长，25°C用于不动杆菌的生物膜形成。使用30°C是因为许多研究使用超过30°C的不动杆菌的最佳生长，这对于获得足够的...

已知不动杆菌会引起医院感染，其人类感染，尤其是在医疗机构中，越来越多的报道1.它广泛存在于医院、医疗机构和食品相关环境中 2,3,4。它可以在包括医院表面（如床栏、床头柜、呼吸机表面和水槽）在内的环境中长期存活4.这种在环境表面的持久性可能是导致不动杆菌4 院内感染的重要因素之一。
生物膜是微生物生命的一种形式，是由活的微生物细胞和来自细胞的细胞外聚合物物质（EPS）组成的微生物基质5。微生物细胞嵌入基质中，通常对环境压力（如热、盐、干燥、抗生素、消毒剂和剪切力）具有很强的抵抗力6,7。
不动杆菌可以在表面上形成生物膜，这表明它可能有助于延长在环境表面（包括医院表面）的存活时间，并增强对抗生素治疗的耐药性8,9。此外，不动杆菌的生物膜形成可能与人类临床结果高度相关8。因此，不动杆菌菌株的生物膜形成性可能是预测环境生存和人类感染的指标之一8,10。
表面附着的生物膜可以被量化和可视化，以评估生物膜的形成性。为了量化表面附着的生物膜，通常用生物膜染色染料（如结晶紫）对生物膜进行染色，并将染料在溶液中洗脱并测量光密度11。生物膜的可视化是评估生物膜形成性的另一种好方法11。与其他技术（如 SEM12,13）相比，使用荧光染料的特异性共聚焦激光扫描显微镜 （CLSM） 可视化方法可能更有助于表征生物膜形态。
可以对生物膜中的活细胞进行计数，以估计生物膜中的活细胞数量11。将嵌入生物膜中的活细胞分离、稀释、铺在琼脂平板上、孵育和计数。由于较高的细胞数可能满足感染剂量，因此可以提供有关生物膜的更详细的信息，例如与细胞数相关的感染电位13,14。
本文介绍了 （1） 量化表面附着的生物膜，（2） 对生物膜中的活细胞进行计数，以及 （3） 使用 Acinetobacter 的 CLSM 可视化生物膜的分步方案。所提出的方案描述了评估 不动杆菌 分离株的生物膜形成性和表征其生物膜的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plate</td>
			<td>SPL</td>
			<td>30096</td>
			<td>Polystyrene 96-well plate</td>
		</tr>
		<tr>
			<td>BHI (Brain Heart Infusion) broth</td>
			<td>Merck KGaA</td>
			<td>1.10493.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar Base Plate</td>
			<td>KisanBio</td>
			<td>MB-B1005-P50</td>
			<td>Growth media for Acinetobacter</td>
		</tr>
		<tr>
			<td>CHROMagar Acinetobacter</td>
			<td>CHROMagar</td>
			<td>AC092</td>
			<td>Selective plate for Acinetobacter</td>
		</tr>
		<tr>
			<td>Crystal violet solution</td>
			<td>Sigma-Aldrich</td>
			<td>V5265</td>
			<td></td>
		</tr>
		<tr>
			<td>Filmtracer LIVE/DEAD biofilm viability kit</td>
			<td>Invitrogen</td>
			<td>L10316</td>
			<td>SYTO9 and propidium iodide</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200 PRO NanoQuant</td>
			<td>Biofilm measurement</td>
		</tr>
		<tr>
			<td>RBC Glass Plating Beads</td>
			<td>RBC</td>
			<td>RG001</td>
			<td>Glass beads</td>
		</tr>
		<tr>
			<td>&#956;-Plate 96 Well Black</td>
			<td>ibidi</td>
			<td>89621</td>
			<td>Microplate intended for CLSM</td>
		</tr>
	</tbody>
</table>

quantification, viability assessment, and visualization strategies for acinetobacter biofilms

不动杆菌 会引起院内感染，其生物膜的形成有助于在医院环境等干燥表面上存活。因此，生物膜定量和可视化是评估 不动杆菌菌 株引起医院感染潜力的重要方法。在微孔板表面形成的生物膜可以根据体积和细胞数量进行量化。生物膜体积可以通过使用结晶紫染色、洗涤、使用乙醇脱色，然后使用酶标仪测量溶解染料来定量。为了量化嵌入生物膜中的细胞数量，使用细胞刮刀将生物膜刮掉，在盐水中收获，在玻璃珠存在下剧烈搅拌，并涂抹在不动杆菌琼脂上。然后，将板在30°C孵育24-42小时。孵育后，计数红色菌落以估计生物膜中的细胞数。这种可行的计数方法也可用于计数混合物种生物膜中的 不动杆菌 细胞。 不动杆菌 生物膜可以使用荧光染料进行可视化。使用设计用于显微分析的市售微孔板来形成生物膜。然后，用SYTO9和碘化丙啶染料染色底表面附着的生物膜，洗涤，然后用共聚焦激光扫描显微镜观察。

按照该方案，最初从厨房表面分离的 不动杆菌 分离株的生物膜在聚苯乙烯96孔板上形成，用结晶紫染色，并将染料溶解在乙醇中并测量生物膜质量（图1）。生物膜的数量因菌株而异，范围从OD 0.04到1.69（图1）。根据 Stepanović 等人 16 建立的标准，除 A. bouvetii 外，所有分离株都形成了生物膜。 A.放射性抗性剂 形成?...

Watch this Scientific Journal Video about A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization at JoVE.com

作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

该方案描述了接种物的制备，使用结晶紫染料在微量滴定板上的生物膜定量，生物膜中的活菌计数以及 不动杆菌生物膜的可视化。

不动杆菌生物膜的定量、活力评估和可视化策略

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...

生物膜由活的微生物细胞组成，很难从表面去除。不动杆菌菌株常见于我们的环境中，引起医院感染，也已知会在表面形成生物膜。因此，我们的目标是开发量化和可视化不动杆菌生物膜的方法。我们的方案提供了一种简单易行的方法来量化和可视化不动杆菌的生物膜。通过使用活菌计数方法量化生物膜内细胞的活菌数。我们的研究结果表明，天然存在的不动杆菌载体菌株具有相当不同的生物膜形成潜力。此外，各种生物膜内将存在明显的形态差异。这为研究不动杆菌菌株之间如此大差异的原因铺平了道路。

quantification-viability-assessment-visualization-strategies-for

Research

JoVE Journal

Biology

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

2.7K 次观看.  Korea Food Research Institute. 生物膜由活的微生物细胞组成，很难从表面去除。不动杆菌菌株常见于我们的环境中，引起医院感染，也已知会在表面形成生物膜。因此，我们的目标是开发量化和可视化不动杆菌生物膜的方法。我们的方案提供了一种简单易行的方法来量化和可视化不动杆菌的生物膜。通过使用活菌计数方法量化生物膜内细胞的活菌数。我们的研究结果表明，天然存在的不动杆菌载体?...

视频: 作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

科研

生物学

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

在大鼠提睾肌的小动脉的可视化和量化的无细胞层

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

使用[18F]FDG微PET / MR成像对小鼠中的棕色和米色脂肪组织进行可视化和定量

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...