Name: basic research in plasma medicine - a throughput approach from liquids to cells
Uploaded: 2017-11-17T12:30:00.000+00:00
Duration: 7 min 37 s
Description: Watch this Scientific Journal Video about Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells at JoVE.com

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

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

微观粒子图像测速技术，微血液流动的速度剖面测量

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

科研

生物工程

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

使用自动工作站进行质谱测量的等离子体样品制备

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

生物化学

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

用于下游无循环DNA应用的标准化液体活检前分析方案

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

癌症研究

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Treating Surfaces with a Cold Atmospheric Pressure Plasma using the COST-Jet

In recent years, non-thermal atmospheric pressure plasmas have been used extensively for surface treatments, in particular, due to their potential in biological applications. However, the scientific results often suffer from reproducibility problems due to unreliable plasma conditions as well as complex treatment procedures. To address this issue and provide a stable and reproducible plasma source, the COST-Jet reference source was developed.
In this work, we propose a detailed protocol to perform reliable and reproducible surface treatments using the COST reference microplasma jet (COST-Jet). Common issues and pitfalls are discussed, as well as the peculiarities of the COST-Jet compared to other devices and its advantageous remote character. A detailed description of both solid and liquid surface treatment is provided. The described methods are versatile and can be adapted for other types of atmospheric pressure plasma devices.

This protocol is presented to characterize the setup, handling, and application of the COST-Jet for the treatment of diverse surfaces such as solids and liquids.

使用成本喷气机用冷大气压力等离子体处理表面

In recent years, non-thermal atmospheric pressure plasmas have been used extensively for surface treatments, in particular, due to their potential in ...

工程学

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

免疫与感染

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

多流灌注生物反应器集成了出口分馏功能，用于动态细胞培养

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

在生物标志物分析临床试验场单次抽血时，准备外周血单核细胞颗粒和血浆

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...