德国联邦教育和研究部 (BMBF 赠款编号03Z22DN11 和 03Z22DN12) 的资金得到了感激的承认。

1. 等离子体物种监测和处理装置
<ol><li>
等离子体物种监测
	<ol>
<li>使用大气压等离子射流和最大两个标准升每分钟饲料气体流量。</li>
		<li>垂直于羽流轴, 在光学发射分光光度计前放置射流, 并使用专用软件记录光电子和波长 (200-1000 nm)。</li>
		<li>混合0.5% 的氮气或氧气, 以干燥或加湿 (5%) 饲料气体。</li>
		<li>对每个气体条件重复光学发射谱。</li>
		<li>使用适合数据图形显示的软件进行分析。</li>
	</ol>
</li>
	<li>
等离子治疗装置
	<ol>
<li>将 jet 修复到计算机驱动的 xyz 表。</li>
		<li>确定井的位置, 并生成一个计算机脚本, 将射流加入到表中, 用适当的处理时间, 在每口井的中心上方。</li>
		<li>添加 104的任何类型的哺乳动物黏附细胞在100µL 的完整细胞培养基 (RPMI1640 10% FCS, 2% 谷氨酰胺, 1% 青霉素/链霉素) 到几个井下的层流罩。</li>
		<li>允许细胞粘附在37° c 的温度下在5% 二氧化碳的湿润气氛中过夜。</li>
		<li>治疗细胞与血浆二十年代在不同的高度 (z-axis) 在250µm 间隔时间。</li>
		<li>在细胞培养基中加入25µL 碘碘化物。</li>
		<li>在显微镜下对细胞进行成像, 以确定不诱发细胞坏死的特定高度, 因为饲料气体将培养基放在细胞顶部。 注: 对于较长的处理时间, 确定在这一特定高度的液体蒸发, 在等离子体上和后的方式, 通过称重板在等离子处理之前和之后确定升 doubledistilled 水的数量需要重建在处理过的井中渗透稳态。</li>
		<li>为 xyz 表准备一个最终协议, 并分别处理时间 (这里: 二十年代、四十年代、六十年代等离子和六十年代气体控制) 和井位, 并在随后的化验中准备了多通道管和储液处理96井板。</li>
	</ol>
</li>
</ol>2. 等离子体处理液体中主要反应组分的分析
<ol><li>
等离子体处理过氧化氢的分析
	<ol>
<li>将100µL 的磷酸盐缓冲盐水 (PBS) 与等离子在平底 96-井板中进行三份处理。</li>
		<li>通过加载100µL 的 PBS 与 10 U 的辣根过氧化物酶和5µM 的过氧化氢检测试剂 (足够的一个) 准备一个主混合; 相应地缩放。</li>
		<li>每个井加95µL 的主配料。 注: 前一步应在干净的工作台下或在与等离子体源分离的房间内进行, 因为环境空气臭氧可以降低检测的灵敏度。</li>
		<li>在一个单独的板块, 准备一个两倍稀释系列过氧化氢在 pbs 与最高浓度100µM 在100µL pbs。</li>
		<li>在含有检测试剂的水井中加入5µL 样品或过氧化氢标准。</li>
		<li>包含检测试剂的井只用于出后的背景减影。</li>
		<li>在室温下在黑暗中孵育板15分钟。</li>
		<li>放置在微读者测量荧光在λex 535 nm 和λem 590 nm。 注: 如果样品中含有高过氧化物浓度, 则需要增加样品稀释量, 或在潜伏期较短时进行读数, 以避免化验的饱和。</li>
		<li>从所有样本中减去空白值, 计算过氧化物标准曲线, 并从该基线曲线中量化未知样品的过氧化物浓度。</li>
	</ol>
</li>
	<li>
等离子体处理亚硝酸根的分析
	<ol>
<li>flatbottom 96well 钢板治疗100µL 的 PBS 和血浆。</li>
		<li>将1µL 的定量试剂加到99µL 的 doubledistilled 水中, 准备一份亚硝酸盐检测溶液的主混合液 (足够一个)。</li>
		<li>添加100µL 每井到一个明确的, flatbottom 96well 板。根据需要扩大油井数量。</li>
		<li>在 doubledistilled 水中制备稀释系列的亚硝酸盐标准。</li>
		<li>在96well 版的主组合中加入10µL 标准或样品。</li>
		<li>在室温下在黑暗中孵育板10分钟。</li>
		<li>添加5µL 亚硝酸盐量化开发商解决方案, 以每井。</li>
		<li>阅读荧光在微读者在λex 365 nm 和λem 450 nm。</li>
		<li>从所有样本中减去空白值, 计算亚硝酸盐标准曲线, 并从该基线曲线中量化未知样品的亚硝酸盐浓度。</li>
	</ol>
</li>
	<li>
等离子体处理超氧化物的分析
	<ol>
<li>在 PBS 中制作氧化细胞色素 (1 毫克/毫升) 和过氧化氢酶 (20 µg/毫升) 的主混合。</li>
		<li>将100µL 的主混合料加到一个清晰的平底96孔板的井中。</li>
		<li>治疗的主混合与血浆的每一次处理条件。</li>
		<li>使用微阅读器读取 550 nm 的吸光度;绘制此数据。</li>
		<li>计算了利用细胞色素 C 的摩尔消光系数和井内液体的光照路径长度生成的超氧化物的量。</li>
	</ol>
</li>
</ol>3. 暴露在等离子体中的细胞的生物反应
<ol><li>
血浆处理细胞的多维读出: 代谢活性
	<ol>
<li>使用层流罩为以下所有过程。</li>
		<li>种子 104细胞 (B16 小鼠黑色素瘤) 在100µL 完全补充细胞培养基每井在平底 96-井板。</li>
		<li>允许细胞黏附在37° c 过夜在恒温环境的孵化器与5% 的二氧化碳。</li>
		<li>使用 xyz 表, 根据预先定义的方案, 单独对井进行等离子体或气体处理, 并将细胞重新放入孵化器20小时。 注: 如需, 在20小时后脱清, 分析细胞外产物;之后添加100µL 的新鲜培养基。</li>
		<li>对每一个井, 添加25µL 细胞培养基含有500µM 天青 (最终浓度100µM);准备三只含天青的井, 在没有细胞的细胞培养培养基中进行背景减法。</li>
		<li>孵化3小时。</li>
		<li>读荧光在λex 535 nm 和λem 590 nm 在一个微的读者。</li>
		<li>从所有样本中减去背景荧光, 并将数据规范化以控制值。</li>
	</ol>
</li>
	<li>
等离子体处理细胞的多维读出: 图像分析
	<ol>
<li>使用3.1.7 的井板, 丢弃上清液。</li>
		<li>添加100µL 的新鲜细胞培养基含有1µg/毫升碘碘化物。</li>
		<li>把盘子放在显微镜下, 用电动的舞台把每个井都想象出来。 注: 成像细节取决于实验问题;在这里, 一个20X 的目标是使用在一个高成像设备, 以读取3x3 的视野, 在每个井在明亮的现场通道, 数字相衬通道, 荧光 (碘碘) 通道, 利用适当的激发光和发射器.</li>
		<li>利用定量图像分析软件, 确定了各视场图像的全数字相位对比图的总胞浆面积。 注意: 如果需要, 分析进一步的参数, 例如, 使用专用污渍的可行和/或死细胞或线粒体活动的数量。如果使用其他污渍比本工作中所描述的, 请确保用于显微镜的荧光染色不光谱重叠的荧光用于后续流式细胞仪实验。</li>
		<li>对数据进行图表, 如果需要的话, 测试与代谢活动所获得的值的相关性。</li>
	</ol>
</li>
	<li>
等离子体处理细胞的多维读出: 流式细胞仪
	<ol>
<li>对于这一分析, 使用3.2.5 的油井板。</li>
		<li>丢弃上清液两次, 用200µL 的 PBS, 含有钙和镁 (分别为0.9 毫米和0.5 毫米)。 注: 在这个阶段修复和/或 permeabilize 细胞的进一步生物学读数不包括在这个贡献, 如染色细胞内抗原或细胞周期分析。</li>
		<li>每井, 添加50µL 的 PBS 与钙和镁含有 50 ng/毫升 anti-mouse 钙单克隆抗体。</li>
		<li>孵育15分钟的孵化器。</li>
		<li>用200µL 完全补充的细胞培养基清洗两次, 并将液体丢弃在盘中。</li>
		<li>每井加100µL 细胞剥离液, 孵化20分钟。</li>
		<li>在悬浮中使用另一组不 B16 单元来设置流细胞采集协议、浇注策略以及探测器的增益和电压。</li>
		<li>将该板装入流式细胞仪, 配有自动取样器用于96井板。</li>
		<li>在前方和侧面散射群中获得至少1000细胞, 通常与活细胞相关。</li>
		<li>使用专用于流式细胞仪分析的软件,. fcs 文件, 以门的利益群体, 并确定平均荧光强度的钙。</li>
		<li>图表数据, 如果需要, 测试与获得的价值的新陈代谢活动, 成像, 和/或氧化剂沉积。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

基础研究是基础, 以了解的功效和机制为基础的等离子医学在前临床研究。基础研究也促进了对新的治疗应用的调查。虽然它是公认的等离子体调解他们的生物效应通过生成的活性氧和氮的物种19, 仍然有三主要挑战的领域。首先, 哪些物种是重要的？通过光学诊断和生物读数调节等离子体的进气成分, 可以部分地回答这一问题。20秒, 在生物目标 (如细胞) 中可以看到什么效果？这至少在某种程度上是用细胞培养实验和一些化验方法解决的。在真核细胞中, 效包括细胞周期阻滞21, 凋亡22, 坏死23, 和皮肤细胞刺激24,25,26, 以及支持或减少细胞运动或新陈代谢活动27,28,29。第三个挑战, 与这些效的影响, 是确定的关键分子, 确定细胞反应等离子体衍生氧化剂, 以解释不同的影响, 经常看到不同的细胞类型。这可以通过学技术30,31,32和/或下调的目标基因使用 siRNA (氧化还原) 信号抑制剂, 抗氧化剂和抗氧化酶, 以及调制等离子体的反应物种输出。33,34,35因此, 需要使用简化的检测协议来测试较大数量的迭代大样本集。
这项研究表明了一个有效的实验工作流程, 从物理到生物学, 等离子体医学研究的有关上述挑战。详细介绍了等离子体源和等离子体产生的工程方面。36,37,38所有这些测试都是在96well 版中进行的, 具有许多优点。例如, 细胞培养清等样品可以很容易地从化验到收集板进行研究, 例如, 通过酶联免疫吸附法测定蛋白质浓度。如果能够直接从96口井读取的科学设备是可用的, 那么这种方法可以将每样样品的成本降到最低, 并 handsontime, 并通过从同一样品中复用不同的化验结果最大化。多通道管的使用还加速了样品处理。在原则上, 这里描述的分析也可以使用井板格式与更大的井径, 虽然这将需要另外的移步入其他管子和船为下游化验。较小的平板类型, 如384well 板, 但是, 不提出的几何适用于生物医学等离子体研究使用大的饲料气体流量的喷气机。具体而言, 不能保证在处理过程中, 只有单个但不相邻的水井受到影响。
液体分析对研究等离子体中的物种沉积是至关重要的。在专家分析中, 不同检测的平行设置可以同时对不同的氧化剂进行 plasmatreated 液体的表征。这种多路复用允许开发 plasmaderived 物种的区分图片。所描述的方法是高度敏感的调查过氧化氢浓度39, 这是经常是必要的, 但并不总是足够的解释等离子体效应。40,41,42生物学上的相关效应也可以用不包括在这里的谷胱甘肽测定的液体来评估。43特定的亚硝酸盐检测是强烈推荐的传统格里斯-分析由于10fold 更高的检测灵敏度 (数据没有显示)。Plasmatreated 液体也可以研究的次氯酸酸的形成使用 DTNB-检测。然而, 以前的研究结果并没有表明该物种与我们的等离子体源的形成。19,39,44等离子体处理完成后, 物种的退化会随着时间的推移而发生。亚硝酸盐对硝酸盐有反应;同时, 过氧化物也会随着时间的推移而消耗。但是, 这些过程需要几个小时。35细胞色素 c 吸收也在几个小时内稳定。因此, 如果在治疗后的前30分钟内发生的过程, 在这里描述的长寿物种的浓度变化是微不足道的。然而, 在调查某些类型的介质 (例如, Dulbecco 氏改良的鹰培养基) 时必须小心, 因为成分可以在1小时内清除多达90% 的过氧化物 (数据未显示), 导致对血浆过氧化物沉积的低估液体.
本文介绍了从细胞面积 (形态学) 到细胞表面标记表达的研究。这些化验的组合可能会显示出有趣的结果。例如, 我们以前曾在 THP1 单核细胞中显示, 在暴露于血浆后, 代谢活动和体细胞数不会以线性方式减少。45相反, 随着血浆处理时间的增加, 细胞被观察到的大小增加, 并有较高的线粒体质量, 从而导致更高的新陈代谢率在 percell 的基础上。从根本上说, 多的读者, 显微镜, 流式细胞仪多信息的细胞反应后, 等离子体治疗。在黑色素瘤细胞中, 我们关注的是细胞毒性作用和钙介导的免疫原性。46原则上, 可以通过这种方法将代谢活动与成像和流式细胞仪结果联系起来, 解决许多其他问题。例如, 细胞分化 (如巨噬细胞极化), 线粒体膜电位, 细胞周期分析, 细胞运动, 生物力学, 或微核形成分析遗传毒性, 也可以研究等离子体处理的细胞。多色流式细胞仪允许在不同的细胞群中同时使用更多的应用。例如, 这包括对信号蛋白磷酸化状态的分析, 如转录因子、mRNA 量化、细胞内细胞因子的测定和/或对单个细胞水平的总还原硫的评估。在进一步发展等离子氧化还原效应的图片以更好地了解当前和将来的等离子体应用方面, 可为每个样本提供更多的生物相关信息。

反应性物种是重要的氧化还原调节因子, 包括异常伤口愈合1和癌症。2重要的是, 这些物种参与了病理学以及它的治疗解决方案。3,4冷物理等离子体是一种电离气体, 可排出多种活性物质。5由于 so-called 低温等离子体的出现, 在体温的6, 冷等离子体可以应用到细胞和组织没有热损害。通过证明等离子设备在临床前和观察中的功效和安全应用, 三在德国获得了医疗器械认证。7特别是在毒性安全方面, 有几项研究表明, 使用第一种装置, 常压氩等离子体射流没有诱变事件。8,9,10其他两个设备是 so-called 介质阻挡放电 (阻挡), 通过不同的原则操作比血浆喷气机。具体而言, 喷气机允许手术刀般的表面和空洞的治疗, 而阻挡放电等离子是有效的治疗较大的, 但相当平坦的组织区域。利用细胞氧化还原信号11, 该技术的目的是利用等离子体产生的反应种来进行生物医学应用。12临床等离子治疗的一个特别有前途的应用是伤口愈合的支持。13,14,15此外, 在动物模型中, 等离子体被证明具有抗癌作用。16,17,18
在验证血浆应用在动物模型, 甚至人类的有效性和安全性之前, 标准化的体外研究需要用等离子设备进行。这些实验对于探索等离子体应用和描绘工作中的机制是必不可少的。此外, 还需要进行基础研究, 以了解反应物种组成的影响和随后的生物效应。这项研究展示了如何将等离子体集成到生物医学研究中, 以更好地理解和控制其对细胞的影响。它描述了对饲料气体成分的调整, 对反应性物种输出的监测, 血浆对液体、细胞和组织的应用, 以及由此产生的化学和生物结果。此外, 还提供了一些例子, 说明等离子体处理和下游生物化验的标准化, 重点放在96井板上的等离子体医学研究上。此方法具有明显的优点: i) 最小化每个条件所需的单元格数、试剂成本和每个样本的实际操作时间;(二) 可方便地设置重复或三份治疗结果的更高精确度的津贴;和 iii) 简化 96-井格式板读写器、成像和流式细胞仪实验的无缝下游检测。

<table style="border-collapse:collapse;width:756pt" width="1008">
	<colgroup>
		<col style="width:385pt" width="513" />
		<col style="width:99pt" width="132" />
		<col style="width:120pt" width="160" />
		<col style="width:152pt" width="203" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">accutase</td>
			<td class="xl20" style="width:99pt" width="132">BioLegend</td>
			<td class="xl20" style="width:120pt" width="160">423201</td>
			<td class="xl21" style="width:152pt" width="203"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">amplex Ultra Red detection kit (includes horse-radish peroxidase and H2O2)</td>
			<td class="xl20" style="width:99pt" width="132">Thermo</td>
			<td class="xl20" style="width:120pt" width="160">A36006</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">argon gas</td>
			<td class="xl20" style="width:99pt" width="132">Air Liquide</td>
			<td class="xl20" style="width:120pt" width="160">N50</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">B16F10 murine melanoma cells</td>
			<td class="xl20" style="width:99pt" width="132">ATCC</td>
			<td class="xl20" style="width:120pt" width="160">CRL-6475</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">catalase</td>
			<td class="xl20" style="width:99pt" width="132">Sigma</td>
			<td class="xl20" style="width:120pt" width="160">C30</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">cell culture incubator</td>
			<td class="xl20" style="width:99pt" width="132">Binder</td>
			<td class="xl20" style="width:120pt" width="160">CB210</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">cell culture plastic</td>
			<td class="xl20" style="width:99pt" width="132">NUNC</td>
			<td class="xl20" style="width:120pt" width="160">156545</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">cytochrome c, oxidized</td>
			<td class="xl20" style="width:99pt" width="132">Sigma</td>
			<td class="xl20" style="width:120pt" width="160">C2037</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">data analysis</td>
			<td class="xl20" style="width:99pt" width="132">GraphPad software</td>
			<td class="xl20" style="width:120pt" width="160">prism 7.03</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">fetal bovine serum</td>
			<td class="xl20" style="width:99pt" width="132">Sigma</td>
			<td class="xl20" style="width:120pt" width="160"></td>
			<td class="xl21">batch-tested</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">fine scale</td>
			<td class="xl20" style="width:99pt" width="132"></td>
			<td class="xl20" style="width:120pt" width="160"></td>
			<td class="xl21">any with resolution of 0.01mg</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">flow cytometer</td>
			<td class="xl20" style="width:99pt" width="132">Beckman-Coulter</td>
			<td class="xl20" style="width:120pt" width="160">CytoFlex S</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">flow cytometry data analysis</td>
			<td class="xl20" style="width:99pt" width="132">Beckman-Coulter</td>
			<td class="xl20" style="width:120pt" width="160">Kaluza 1.5a</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">Glutamine</td>
			<td class="xl20" style="width:99pt" width="132">Sigma</td>
			<td class="xl20" style="width:120pt" width="160">G7513</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">imaging quantification software</td>
			<td class="xl20" style="width:99pt" width="132">PerkinElmer</td>
			<td class="xl20" style="width:120pt" width="160">Harmony 4.5</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">laminar flow hood</td>
			<td class="xl20" style="width:99pt" width="132">Thermo</td>
			<td class="xl20" style="width:120pt" width="160">Maxisafe 2020</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">mass flow controller</td>
			<td class="xl20" style="width:99pt" width="132">MKS</td>
			<td class="xl20" style="width:120pt" width="160">G-series</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">Measure-iT nitrite detection kit</td>
			<td class="xl20" style="width:99pt" width="132">Thermo</td>
			<td class="xl20" style="width:120pt" width="160">M36051</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">microscope</td>
			<td class="xl20" style="width:99pt" width="132">PerkinElmer</td>
			<td class="xl20" style="width:120pt" width="160">Operetta CLS</td>
			<td class="xl21"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl17" height="42" style="width:385pt;height:31.5pt" width="513">optical emssion spectroscopy</td>
			<td class="xl20" style="width:99pt" width="132">Avantes</td>
			<td class="xl20" style="width:120pt" width="160">AvaSpec-DDDD-2-USB2</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">penicilin/streptomycin</td>
			<td class="xl20" style="width:99pt" width="132">Thermo</td>
			<td class="xl20" style="width:120pt" width="160">15140122</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">pipettes</td>
			<td class="xl20" style="width:99pt" width="132">Eppendorf/Brand</td>
			<td class="xl20" style="width:120pt" width="160">single/multi chanel</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">plate reader</td>
			<td class="xl20" style="width:99pt" width="132">Tecan</td>
			<td class="xl20" style="width:120pt" width="160">M200pro</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">propidium iodide</td>
			<td class="xl20" style="width:99pt" width="132">BioLegend</td>
			<td class="xl20" style="width:120pt" width="160">421301</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">resazurin</td>
			<td class="xl20" style="width:99pt" width="132">VWR</td>
			<td class="xl20" style="width:120pt" width="160">B21187.06</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">RPMI1640 cell culture media</td>
			<td class="xl20" style="width:99pt" width="132">PanbioTech</td>
			<td class="xl20" style="width:120pt" width="160">P04-16500</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:385pt;height:15.75pt" width="513">xyz table</td>
			<td class="xl20" style="width:99pt" width="132">CNC step</td>
			<td class="xl20" style="width:120pt" width="160">HIGH-Z S-400/T&#160;</td>
			<td class="xl21"></td>
		</tr>
	</tbody>
</table>

basic research in plasma medicine - a throughput approach from liquids to cells

在等离子体医学中, 与脊椎动物系统相近的电离气体被用于细胞和组织。冷等离子体产生的反应性物种已知的氧化还原调节的生物过程中的健康和疾病。临床和临床证据表明, 血浆治疗对皮肤慢性溃疡愈合有有益的作用。其他新兴话题, 如血浆癌症治疗, 正在受到越来越多的关注。等离子体医学研究需要在物理, 化学和生物医学方面的跨学科的专业知识。等离子体研究的一个目标是在各种特定应用中表征等离子体处理的细胞。这包括细胞计数和活力, 细胞氧化, 线粒体活动, 细胞毒性和死亡模式, 细胞周期分析, 细胞表面标记表达, 细胞因子释放。本研究描述了等离子体生物医学研究所需的基本设备和工作流程。描述了常压氩等离子体射流的正确操作, 具体监测了其基本发射谱和馈气设置, 以调节反应的种类输出。使用高精度 xyz 表和计算机软件, 在微米精度的96孔板的腔内, 以毫秒精度盘旋, 以达到最大再现性。对氧化还原活性分子的液相分析进行了下游测定, 并对靶细胞进行了等离子体处理。具体来说, 黑色素瘤细胞的分析在一个有效的序列不同的连续化验, 但使用相同的细胞: 代谢活动的测量, 总细胞面积, 和表面标记表达的钙, 一个重要的分子肿瘤细胞免疫细胞死亡。这些化验结果从一个单一的板块中获取关于等离子体效应的富含内容的生物信息。总之, 本研究描述了等离子体医学研究的基本步骤和协议。

在这项研究中, 一个简化的工作流程被描述为医学研究对血浆的作用。本文采用的多学科方法分析了等离子体射流的基本光学发射剖面、液体中的主要活性成分以及等离子体处理后细胞的生物反应 (图 1)。
要执行此工作流, 需要一些组件来正确设置等离子体源 (图 2)。不同的气体 (这里主要氩, 氧气和氮气) 被提供了并且由几个质量流量控制器控制。一个中央面板被用来对质量流量控制器进行数字调整, 以确定进料气体通量。为了产生特定的饲料气体成分, 气体是物理混合使用的一个小组的阀门, 组合气体从几个质量流量控制器 (图 2a)。等离子体源被打开, 并且血浆流出物在一个 USB 分光光度计 (图 2B) 前面设置了以200毫微米的光学范围到1000毫微米。为处理液体和细胞, 一个有效的工作流程用96井板描述。多好的菜肴是使用一个塑料框架, 固定在基板上, 插入与其印迹孔匹配 (图 2C)。整个安装被放置在层流罩 (图 2D) 下。此设置包括一个 xyz 表, 其中等离子射流的 hand-held 片安装。马达控制元件可以位于工作台的外面, 如果后者有足够大的电缆端口从和到 xyz 表。重要的是, 该表是由计算机控制的, 可以编程, 在每个井的中心上空盘旋的喷气机与微米精度的期望量的时间。此外, 开始位置 (如在我们的设置, 以及 A1) 是自由选择的 (图 2E)。与塑料板持有人一起, 这允许一个可再生的等离子处理与任何日常的变化完全有关的设置的液体和生物实验。
在不同的进气条件 (图 3A) 中, 使用光学发射光谱仪跟踪与反应等离子体元件相连接的不同峰值。例如, 第二个正系统的氮与峰值从 330 nm 到 380 nm 代表活性氮物种, 峰值在 309 nm 代表羟基自由基 (箭头在图 3A)。与单独的氩气相比, 氮的存在增加了与氮气的混合物入饲料气体, 而氧气或湿气的加法减少了或减少了它, 分别。相反, 羟基自由基的存在随着氧或氮的增加而降低, 但如果用湿化氩作为饲料气体, 则明显增大。对于液体的等离子体处理, 首先确定氩气和氩等离子体引起的蒸发 (图 3B)。重要的是, 这两种情况都没有产生相似的结果, 因为等离子体也会对温度产生影响。根据羟基自由基的发射光谱结果, 过氧化氢沉积物随氧或氮外加剂的增加而明显降低, 但随加湿的进气量增大 (图 3C)。此外, 与氩等离子体处理的液体 (图 3D) 相比, 在饲料气体中添加氮气导致亚硝酸盐浓度明显增高。这一工作流程也可能被用来研究等离子体对不同成分的液体的影响。例如, 过氧化氢浓度似乎是独立的存在胎儿小牛血清在 PBS 和 RPMI1640 细胞培养基 (图 3E)。在相同的样本中, 血清中亚硝酸盐浓度在 PBS 和细胞培养基中的存在降低, 与含有对应物的 non-serum (图 3F) 相比较。大多数超氧化物是在干氩气条件下产生的氧和/或氮外加剂显著淬火超氧化物生成, 除湿式氩氧等离子体 (图 3G)。
对于井皿中细胞的等离子体处理, 前一天被播种的含有细胞的平板被从孵化器中取出并添加到塑料支架上。采用程序化处理模式, 对蒸发进行补偿, 并将钢板放回孵化器20小时. 注意, 在这种孵化后, 细胞培养清可能被收集到一个新的, 空的96孔板和存储在-80° c 用于评估感兴趣的蛋白质。接下来, 天青被添加到每个井的细胞中。荧光天青到荧光卤的营业额只由活性的生成酶产生, 并与整体代谢活性相关 (图 4A)。荧光强度与平板的视觉感知相似, 并显示长时间血浆治疗的细胞毒作用 (图 4B)。湿气条件比干气条件更有害。板中的相同细胞被用于进一步的下游化验。显微镜下研究了平板中的细胞 (图 4C)。利用成像软件, 检查了板的几个井进行定性比较 (图 4D)。软件允许对每个井中所获得的视图字段中的单元格所覆盖的总面积进行量化, 结果数据被规范化为各自的控件 (图 4E)。在等离子体处理样品中, 特别是在湿化饲料气体条件下, 总细胞面积减少。成像后, 细胞洗净, 染色 anti-mouse 钙抗体, 分离, 并分析流式细胞仪 (图 4F)。平均荧光强度的钙染色在可行的细胞 (图 4G) 是计算和比较之间的样本 (图 4H)。等离子体治疗诱导小鼠黑色素瘤细胞表面钙上调, 与干饲料气条件下的等离子体处理时间相对应。对于湿化饲料气体的条件, 二十年代和四十年代的治疗导致更强的钙暴露比更致命的六十年代治疗时间。这表明, 对于钙暴露在细胞中的氧化剂数量, 有一个非线性的调节。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56331/56331fig1.jpg"> 图 1: 等离子体医学研究的工作流程从物理到生物学.对常压氩等离子体射流的反应种输出进行了调节。利用光谱学对等离子体气相中的选定分子进行监测。等离子体用于处理液体和研究氧化剂沉积。在板培养的细胞是等离子体处理的, 并进行生物学读数。<a href="//cloudflare.jove.com/files/ftp_upload/56331/56331fig1large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56331/56331fig2.jpg"> 图 2: 等离子研究台的设置.(A) 不锈钢管道中的不同气体被驱动成几个通过中央面板控制的质量流量控制器。单独的饲料气体组成混合后, 使用一个面板的阀门。(B) 可以使用光学发射光谱对等离子体中的一些活性成分进行监测, 以研究各种气体成分之间的差异。(C) 用于高通量等离子体研究, 使用96井板。为了确保可编程和自动等离子处理的恒定起点, 该板被添加到一个框架, 保证相同的绝对位置的板材从每天。(D) 等离子射流固定在计算机控制的 xyz 表中, 位于层流罩下。所有96确切的位置和血浆的居住时间在每个井被写入程序文件引导血浆来源的运动。等离子体射流装置的主壳体以及马达控制器位于接近附近。(E) 96 井板的自动等离子处理。<a href="//cloudflare.jove.com/files/ftp_upload/56331/56331fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56331/56331fig3.jpg"> 图 3: 等离子射流和液体分析.(A) 显示不同的进气条件的光发射谱。第二正系统的氮 (330 nm 至 380 nm) 增加了氮的混合物, 没有在氧气混合的情况下, 并显着减少湿化氩 (左手板)。309 nm (箭头) 的羟自由基峰值在氮气和氧气条件下下降, 但与氩气相比, 湿化氩明显增加。(B) 液体的气体暴露导致蒸发。在使用等离子和氩气处理的情况下, 在96well 板上的每井蒸发量是通过在处理前和治疗后用一个精细的刻度来确定的。与氩 gastreated 样品相比, plasmatreated 样品中的蒸发更高。(C) plasmatreated 液体中过氧化氢的生成与 (A) 中羟自由基的 309 nm 峰值有一定程度的相关。加湿 (5%) 氩等离子体增加过氧化物浓度约4fold。(D) 在将氮气添加到进料气中时, 亚硝酸盐升高, 而这种作用随加湿氩等离子体而增加。氧的加入减少了亚硝酸盐的生成, 但不显著。(E, F)过氧化氢和亚硝酸盐浓度显示在不同类型的液体, 即 RPMI1640 细胞培养基与 (R10F) 和没有 (R0F) 以及 PBS 与和没有胎儿小牛血清 (FCS)。过氧化物水平在不同培养基 (E) 之间没有差别, 而血清中亚硝酸盐水平则明显降低。(G) 干氩气进气的超氧化物产量最高;氮气、氧气或加湿氩的混合物使液体中的超氧化物沉积物更低。显示的数据 (B-G) 是两到三实验的平均值 + SEM。进行了统计分析 (C, D), 采用 one-way 分析的方差比较了整组的干湿进气条件对各自控制的影响 (** p < 0.01; ** < 0.001)。另外, 用全组平均值和 t 检验 (## < 0.001) 对氩气等离子体条件进行了比较。进一步的统计分析 (F) 采用 t 检验比较了样品中的每种等离子体处理和培养基条件的值与 fcs 和无 fcs (* p < 0.05; ** < 0.01);此外, 使用 t 检验 (# p < 0.05; ## < 0.001), 在每个血浆治疗时间内对 fcs 或非 fcs 的溶液进行了比较。<a href="//cloudflare.jove.com/files/ftp_upload/56331/56331fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56331/56331fig4.jpg"> 图 4: 等离子体处理及其对细胞的影响(A) 细胞暴露于血浆, 天青在20小时后增加, 以评估代谢活动。(B) 与气体控制相比, plasmatreated 样品的活性显著降低。气体加湿会明显减少, 而氧气和/或氮气的增加会导致干燥和潮湿的氩气条件的毒性。(C) 添加含有碘碘化物 (PI) 的培养基。(D) 概述了带有数字相衬 (橙色) 的板的井, 该板块代表每个细胞和 PI (绿色) 的胞浆分数来识别死细胞。成像工具允许定量的总面积在视野内的每个井覆盖的细胞。(F) 细胞随后用抗体或其他细胞标记进行冲洗和孵育, 以利用流式细胞仪表征生物等离子体的作用。(G) 采用了浇注策略, 并对样品进行了标记分析和比较。显示的数据 (B、E、H) 表示三独立实验的一个代表的平均 + SEM。缩放条形图 = 200 µm (D)。统计比较是使用 two-way 分析方差 (B, H) 进行比较, 对每个气体条件, 每个等离子体处理其各自的气体控制 (* p < 0.05; ** < 0.01; ** p < 0.001)。另外, 对氮气和/或氧外加剂的每个条件的值分别与氩气等离子体的值进行了比较 (two-way 的方差分析; < 0.001)。<a href="//cloudflare.jove.com/files/ftp_upload/56331/56331fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells at JoVE.com

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

一个高通量的冷物理等离子体处理协议的液体和细胞显示。它包括设置不同的等离子体点火用气体成分, 测量等离子体的发射光谱, 以及随后对等离子体处理后的液体和细胞活性进行分析。

等离子体医学基础研究--从液体到细胞的吞吐量方法

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

basic-research-plasma-medicine-throughput-approach-from-liquids-to

Research

JoVE Journal

Environment

12.7K 次观看.  Leibniz-Institute for Plasma Science and Technology. 一个高通量的冷物理等离子体处理协议的液体和细胞显示。它包括设置不同的等离子体点火用气体成分, 测量等离子体的发射光谱, 以及随后对等离子体处理后的液体和细胞活性进行分析。

视频: Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

科研

环境科学

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

设计一个城市径流研究设施的建设

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

现代树轮研究技术的角度 - 如何克服Dendroecological和木解剖挑战

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

水生微生物Metaproteomics工作流程:从细胞对胰蛋白酶肽适合串联质谱为基础的分析

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

管理和检测蛋白标记的节肢动物的扩散研究

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

木质纤维素生物质预处理低成本离子液体

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal behavior questions. The protocol described here provides a simple, reproducible, and controlled method to manipulate DO to study behavioral response in aquatic organisms resulting from hypoxic and anoxic conditions. While performing degasification of water with nitrogen is commonly used in laboratory settings, no explicit method for ecological (aquatic) application exists in the literature, and this protocol is the first to describe a protocol to degasify water to observe organismal response. This technique and protocol were developed for direct application for aquatic macroinvertebrates; however, small fish, amphibians, and other aquatic vertebrates could be easily substituted. It allows for easy manipulation of DO levels ranging from 2 mg/L to 11 mg/L with stability for up to a 5 min animal-observation period. Beyond a 5 min observation period water temperatures began to rise, and at 10 min DO levels became too unstable to maintain. The protocol is scalable to the study organism, reproducible, and reliable, allowing for rapid implementation into introductory teaching labs and high-level research applications. The expected results of this technique should relate dissolved oxygen changes to behavioral responses of organisms.

This article describes a simple and reproducible protocol to manipulate dissolved oxygen conditions in a laboratory setting for animal behavior studies. This protocol may be used in both teaching and research laboratory settings to evaluate organismal response of macroinvertebrates, fishes, or amphibians to changes in dissolved oxygen concentration.

一个简单的方法来操作的动物行为观察溶解氧

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

使用 DMAv为环境应用 DMMTAv和 DMDTAv的准备: 合成、纯化和确认

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

等离子体医学基础研究--从液体到细胞的吞吐量方法

摘要

探索更多视频

此视频中的章节

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

设计一个城市径流研究设施的建设

全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统

现代树轮研究技术的角度 - 如何克服Dendroecological和木解剖挑战

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

水生微生物Metaproteomics工作流程:从细胞对胰蛋白酶肽适合串联质谱为基础的分析

管理和检测蛋白标记的节肢动物的扩散研究

木质纤维素生物质预处理低成本离子液体

一个简单的方法来操作的动物行为观察溶解氧

使用 DMA^v为环境应用 DMMTA^v和 DMDTA^v的准备: 合成、纯化和确认

等离子体医学基础研究--从液体到细胞的吞吐量方法

摘要

探索更多视频

此视频中的章节

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

设计一个城市径流研究设施的建设

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

现代树轮研究技术的角度 - 如何克服Dendroecological和木解剖挑战

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

水生微生物Metaproteomics工作流程:从细胞对胰蛋白酶肽适合串联质谱为基础的分析

管理和检测蛋白标记的节肢动物的扩散研究

木质纤维素生物质预处理低成本离子液体

一个简单的方法来操作的动物行为观察溶解氧

使用 DMAv为环境应用 DMMTAv和 DMDTAv的准备: 合成、纯化和确认

全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统

使用 DMA^v为环境应用 DMMTA^v和 DMDTA^v的准备: 合成、纯化和确认