这项工作得到了新疆维吾尔自治区重点研发项目的支持（2017B03014，2017B03014-1，2017B03014-2，2017B03014-3）。

1. 暴露前的准备
<ol><li>选择同一天出生的受精卵进行暴露测试。</li>
	<li>选择重量相似的鸡蛋。每个受精卵大约为10-12克。</li>
	<li>完全清洁外部粪便和其他碎屑中的所有受精卵。</li>
	<li>用抗生素溶液（青霉素和链霉素，1：1000，室温）对每个预孵化受精卵和要使用的卵子（选择壳形相似的卵子，尤其是蛋尖）进行消毒。用75%的乙醇对孵化器进行消毒。</li>
	<li>用牙科钻头的钝端打开鸡蛋，将蛋壳放在尖端以供进一步使用。在转移受精卵之前，将卵子中的内容倒出来。这是为了保持蛋壳的水分。鸡蛋的开口直径约为3厘米。 注意：为了减少对卵胚的损害，使用牙科钻头打开卵子的钝端，使裂纹尽可能光滑。</li>
	<li>绝育后，将受精卵放在 38 °C 孵化器中，湿度为 60%，为 24-48 小时。确保鸡蛋的钝端朝上。</li>
	<li>在受精卵的孵育过程中，对随后在灭菌锅中实验所需的工具进行消毒。这些工具包括塑料包装、烧嘴、无菌水、移液器尖、手术直剪刀、钳子和勺子。 注意：使用耐温度足够高的薄膜，以避免高温灭菌问题。</li>
</ol>2. 在没有壳的情况下孵化鸡蛋
<ol><li>将预孵化受精卵从孵化器转移到干净的长凳上，平放在容器上，使其稳定约1-2分钟。</li>
	<li>使用剪刀（12.5 厘米手术直剪刀）在预孵化受精卵的中轴戳一个小孔（直径 3 毫米），并切开 1-2 厘米的小开口。小心地将受精卵的蛋清和蛋黄转移到切蛋壳中。 注意：用剪刀切小开口时，避免触摸蛋黄。</li>
	<li>通过移液器将具有三粒大小（100、200 和 500 nm）的不同质量（0.1、0.2 和 0.3 毫克）微塑料的控制溶液（无 MP）和暴露溶液添加到鸡蛋中。同时，用1mL注射器加入1滴青霉素和1滴链霉素。</li>
	<li>用消毒膜盖住蛋壳的开口（第 1.6 步）。</li>
	<li>根据第2.1-2.4步，治疗所有受精卵。</li>
	<li>将移植的胚胎放入湿度为 60% 的 38°C 孵化器中，以备不时之需。在这个实验中，使用±30°的卵子旋转角度。每小时转一次鸡蛋。 注：转移应尽可能快，这需要在早期阶段进行更多的实践。</li>
</ol>3. 样品收集
<ol><li>经过七天的培养，从蛋黄中取出肉眼观察到的发育良好的胚胎，然后用磷酸盐缓冲溶液（PBS）清洗。</li>
	<li>用吸水纸干燥清洁胚胎外的剩余溶液，并在干净的培养皿中称重。</li>
	<li>打开整个胸腔，用针鼻钳将肝脏和心脏从内脏中分离出来，并在清理后立即放入 1.5 mL 离心机管中。</li>
	<li>快速记录电子天平上的重量并计算肝功能指标（HIS = 肝脏重量/体重 x 100）。测量胸骨和身体的长度。</li>
	<li>基于上述指标，评估议员对胚胎发育的影响。 注：这里的胚胎质量是指蛋黄去除的质量。</li>
</ol>4. 数据分析
<ol><li>以平均±标准误差 （SEM） 的形式报告实验数据。</li>
	<li>使用方差的单因子分析来比较多个样本组的方法。显著差值为 α = 0.05。</li>
</ol>

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作者没有什么可透露的。所有作者都宣称，他们不知道竞争的经济利益或个人关系，这似乎会影响本文的工作。

本文通过检测基本发育指标，为评估胚胎发育提供了有效的实验方案。然而，这个实验还是有一些局限性的。 
首先，由于无壳孵化，孵化后期胚胎的死亡率较高。在实验过程中，有人为的无法控制的因素，如正常蛋白质比的破坏。我们限制胚胎的暴露时间，以确保实验的准确性。胚胎毒性的研究只能在胚胎发育的早期和中期进行。其次，对胚胎发育的议员们的研究只在基本?...

2015年塑料废物的产量约为6300万吨，其中十分之一被回收利用，其余被焚烧或埋在地下。据估计，到2050年1月1日，约有12，000吨塑料废物将被埋入地下。随着国际社会对塑料废物的关注，汤普森于2004年首次提出了微塑料的概念。微塑料（MP）是指颗粒直径小于5毫米的小颗粒塑料。目前，研究人员已经探测到，在各大洲、大西洋群岛、内陆湖泊、北极和深海栖息地的海岸线上，议员无处不在。因此，更多的研究人员开始研究议员的环境危害。
生物体可以在环境中摄入议员。在全球233种海洋生物的消化道中发现了MPS（包括100%龟类、36%海豹物种、59%鲸鱼物种、59%海鸟物种、92种海鱼和6种无脊椎动物）8种。此外，议员们可能会阻止生物体的消化系统，积累和迁移在他们的波比9。研究发现，议员可以通过食物链转移，他们的摄入量因栖息地、生长阶段、喂养习惯和食物来源的变化而异。一些研究人员报告说，在海鸟11号的粪便中存在议员，这意味着海鸟充当了议员的载体。此外，摄入议员会影响某些生物体的健康。例如，MP可以纠缠在胃肠道，从而增加鲸目动物的死亡12。
仅议员就对生物体有毒性影响，对具有其他污染物的生物体有联合毒性影响。摄入与环境有关的塑料碎片浓度可能会干扰成年鱼13的内分泌系统功能。微塑料的大小是影响生物体吸收和积累的重要因素之一。小尺寸塑料，特别是纳米大小的塑料，容易与高毒性16、17、18、19等高毒性的细胞和生物体相互作用。虽然纳米颗粒大小的微塑料对生物体的有害影响超过了目前的研究水平，但检测和量化大小小于几微米的微塑料，特别是环境中的亚微子/纳米塑料，仍然是一项巨大的挑战。此外，纳米塑料也对胚胎有一定的影响。聚苯乙烯可以通过调节蛋白质和基因特征20来破坏海胆胚胎的发育。
为了探讨议员对生物体的潜在影响，我们进行了这项研究。由于鸟类胚胎和人类胚胎的相似性，它们通常用于发育生物学研究21，包括血管生成和抗血管生成、组织工程、生物材料植入物和脑肿瘤22、23、24。鸟类胚胎具有成本低、培养周期短、操作方便等优点。因此，在这项研究中，我们选择了生长周期短的胚胎作为实验动物。同时，我们可以通过无蛋壳孵化技术直接观察胚胎发育阶段暴露在 MP 的卵壳胚胎的形态变化。使用的实验材料是聚丙烯（PP）和聚苯乙烯（PS）。由于PP和PS27占全世界沉积物和水体中聚合物类型的最大比例，从捕获的海洋生物中提取的最常见的聚合物类型是乙烯和丙烯28。这个实验协议描述了对议员们对暴露在议员面前的胚胎的毒理学影响进行视觉评估的全过程。我们可以很容易地扩展这种方法来检查其他污染物对其他动物胚胎发育的毒性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Multi sample tissue grinder</td>
			<td>Shanghai Jingxin Industrial Development Co., Ltd.</td>
			<td>Tissuelyser-24</td>
			<td>Grind large-sized plastics into small-sized ones at low temperature</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>OHAUS corporation</td>
			<td>PR Series Precision</td>
			<td>Used for weighing</td>
		</tr>
		<tr>
			<td>Fertilized quail eggs</td>
			<td>Guangzhou Cangmu Agricultural Development Co., Ltd.</td>
			<td></td>
			<td>Quail eggs for hatching without shell</td>
		</tr>
		<tr>
			<td>Fluorescent polypropylene particles</td>
			<td>Foshan Juliang Optical Material Co., Ltd.</td>
			<td></td>
			<td>Types of plastics selected for the experiment</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Shandong, Bangda Incubation Equipment Co., Ltd.</td>
			<td>264 pc</td>
			<td>Provide a place for embryo growth and development</td>
		</tr>
		<tr>
			<td>Nanometer-scale polystyrene microspheres</td>
			<td>Xi&#8217;an Ruixi Biological Technology Co., Ltd.</td>
			<td>100 nm, 200 nm, 500 nm</td>
			<td>Types of plastics selected for the experiment</td>
		</tr>
		<tr>
			<td>Steel ruler</td>
			<td>Deli Group</td>
			<td>20 cm</td>
			<td>Used to measure&#160; length</td>
		</tr>
		<tr>
			<td>Vertical heating pressure steam sterilizer</td>
			<td>Shanghai Shenan Medical Instrument Factory</td>
			<td>LDZM-80KCS-II</td>
			<td>Sterilize the experimental articles</td>
		</tr>
	</tbody>
</table>

ecotoxicological effects of microplastics on bird embryo development by hatching without eggshell

微塑料是一种新兴的全球污染物类型，由于动物组织和器官的吸收和转移，对动物的健康构成巨大威胁。微塑料对鸟类胚胎发育的生态毒性影响尚不清楚。鸟蛋是一个完整的发育和营养系统，整个胚胎发育发生在蛋壳中。因此，在微塑料等污染物的压力下，鸟类胚胎发育的直接记录受到传统孵化中不透明蛋壳的极大限制。在这项研究中，微塑料对卵壳发育的影响通过没有蛋壳的孵化进行视觉监测。主要步骤包括受精卵的清洁和消毒、暴露前的孵化、暴露后的短期孵化以及样品提取。结果表明，与对照组相比，微塑料暴露组的湿重和体长呈统计差异，整个暴露组的肝比例显著增加。此外，我们评估了影响孵化的外部因素：温度、湿度、卵子旋转角度和其他条件。这种实验方法为微塑料的生态毒理学提供了宝贵的信息，是研究污染物对胚胎发育的不利影响的新方法。

在实验数据分析中，比较了湿重、体长、胸骨长度以及对照组与6个实验组肝病指数的变化，从宏观角度测量和反映了胚胎的生长发育。我们在每组中检测到六个正常的胚胎。每个胚胎都达到了所需的汉堡和汉密尔顿（HH）阶段。
在 图1中，我们把预孵化的受精卵含量转移到半球蛋壳中，并把它们放入孵化器中。然后，我们记录了胚胎在潜伏期中期的发育...

Watch this Scientific Journal Video about Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell at JoVE.com

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

本文介绍了一种无需使用蛋壳进行微塑料等颗粒污染物毒理学研究的孵化方法。

微塑料对鸟胚胎发育的生态毒性影响，无需蛋壳孵化

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues ...

这个实验将包括四个部分。暴露前准备，孵化无壳卵，孵化过程和样品采集，并取得具有代表性的结果。请观看以下视频，介绍具体的实验过程。在暴露试验中，为实验选择了同一批受精卵。选择大小相近的卵子以减少实验错误。同时，所有受精的鸡蛋和卵子应完全清洁，因为其壳上会有粪便和其他物质。之后，预先孵化的，受精的卵子，和要使用的鸡蛋消毒青霉素和霉素放置时，你需要注意顶部朝下，然后钝端朝上。视频中没有显示鸡蛋的清洁过程。下一步是蛋壳接地的过程。这是准备中最重要的一步。吟唱的蛋壳是用牙科钻头切开的中心蛋大小，但不能打破。试图使切口的断裂表面足够光滑，然后用75%的Acinol溶液对孵化器进行消毒。将受精卵放入湿度为 60% 和 38 的孵化器中

ecotoxicological-effects-microplastics-on-bird-embryo-development

Research

JoVE Journal

Environment

4.3K 次观看.  Chinese Academy of Sciences. 这个实验将包括四个部分。暴露前准备，孵化无壳卵，孵化过程和样品采集，并取得具有代表性的结果。请观看以下视频，介绍具体的实验过程。在暴露试验中，为实验选择了同一批受精卵。选择大小相近的卵子以减少实验错误。同时，所有受精的鸡蛋和卵子应完全清洁，因为其壳上会有粪便和其他物质。之后，预先孵化的，受精的卵子，和要使用的鸡蛋消毒青霉素和霉素?...

视频: Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

推挽议定书蜜蜂会影响家禽的殖民化巢箱

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

科研

环境科学

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

对水生生命值评价悬浮泥沙影响的流通系统曝光

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

生物毒理学方法与海洋细菌<em&gt;鳗弧菌</em&gt;评估环境污染物的急性毒性

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

真菌 DNA 分析用职业空气标本的收集与提取

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

3维 (3 d) 印制的高吞吐量斑马鱼胚胎排列模板

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the deep ocean. Roles of minerals are critical in many hydrothermal organic geochemical processes. Traditional hydrothermal methodology, which includes using reactors made of gold, titanium, platinum, or stainless-steel, is usually associated with the high cost or undesired metal catalytic effects. Recently, there is a growing tendency for using the cost-effective and inert quartz or fused silica glass tubes in hydrothermal experiments. Here, we provide a protocol for carrying out organic-mineral hydrothermal experiments in silica tubes, and we describe the essential steps in the sample preparation, experimental setup, products separation, and quantitative analysis. We also demonstrate an experiment using a model organic compound, nitrobenzene, to show the effect of an iron-containing mineral, magnetite, on its degradation under a specific hydrothermal condition. This technique can be applied to study complex organic-mineral hydrothermal interactions in a relatively simple laboratory system.

Earth-abundant minerals play important roles in the natural hydrothermal systems. Here, we describe a reliable and cost-effective method for the experimental investigation of organic-mineral interactions under hydrothermal conditions.

研究矿物对有机热液转化影响的实验协议

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

在体外显示砷对皮肤细胞迁移影响的划痕测定

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

用于扫描电子显微镜的胚胎、蛋壳和真菌培养

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...