Name: method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro
Uploaded: 2015-03-21T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1.光学校正:测量光输出<ol><li>为了制备所述光递送装置，连接宽带光源（ 例如 ，氙或钨灯），以适当的电源。定位在光源的前面的准直透镜，以产生的光的准直光束。通过液体热过滤通过的光以从光束去除大部分的热量。根据不同的应用，聚焦准直光束上的入口孔的液体光导的，这提供了更灵活的递送的光的（ 例如 ，到一个培养箱）。 </li><li>在液体光导的另一端，定位一个第二准直透镜，然后对干扰和中性密度滤光片的支架。检查这种安排产生光所需要的波段和强度的均匀照明的地方。 注意:在光导和试样面的端部之间的距离可能需要根据改变必须被照亮，但面积记住作为从光导增大端部的距离，光的强度将减小。在本研究中，这个距离​​是14厘米并产生一个光束截面均匀地照射一个3x3阱区域上的96孔平板。 </li><li>保证从灯和相关的光学组件，杂散光无法到达的试样。 </li><li>放水冷却器用于液体热过滤，并确保存在通过过滤器夹套交换的水。开启灯电源并等待至少5分钟，对宽带光源来稳定。注意:使用水冷却器的需要，以防止过度加热的装置。 </li><li>选择的窄带干扰滤波器（通常由它们的中心波长，峰值透射率和半峰全宽（FWHM）带宽描述），以产生照明的期望的波段。注意:在目前的研究中使用的窄带干扰滤波器分别为442纳米，550纳米，671纳米和810纳米。 </li><li>测量由灯在平面，其中试样是在治疗过程中被定位产生的光。使用连接到一个合适的分光校准的辐射探测器（余弦集电极）测量光，根据制造商的说明使用正当性的软件。 </li><li>使用中性密度滤光片，直到获得所需的输出来调节光的强度。注意:在本研究中，光通过每个干涉滤光器传递的强度被调整到相等的量子输出在各四个治疗波段的。要定期检查校准，因为灯的输出如有变更，这是非常重要的。 注:按照该装置的设置后，所述细胞是准备被照射图1是在目前的研究中使用的光传送装置的表示。 </li></ol><img alt="图1"SRC ="/文件/ ftp_upload / 52221 / 52221fig1.jpg"/&gt; 在光传输设备。画报图1.图像的光电源，氙灯住房，准直透镜，水过滤器，入口孔，液体光导，第二准直透镜，过滤器支架，处理框架和磨砂黑卡。需要注意的是窄带波长和强度的过滤器未示出。 2.细胞的制备<ol><li>所描述的R / NIR-LT递送方法可以适用于任何类型的细胞或体外模型系统;细胞培养等下面的说明是一般情况下，使用成熟的技术，文化嗜铬细胞瘤（PC12），穆勒（RMC1）和原发性视网膜混合细胞。 </li><li>此前细胞接种于光治疗和氧化应激试验，文化永生化细胞在其包含适当的补充（ 例如 ，FBS，抗生素）的T75 FLAS各自的生长介质KS，直到70-80％汇合。 </li><li>分离永生化细胞从T75烧瓶，使用适合于细胞类型的方法进行测试如 。，胰蛋白酶。如果需要的话，前继续进行细胞的96孔测定板的播种，涂层测定板孔用为10μg/ ml的聚-L-赖氨酸1小时（ 例如 ，对于PC12细胞）或10微克/ ml的聚-L-赖氨酸为1H随后的O / N培养用为10μg/ ml的层粘连蛋白（ 例如 ，用于混合的视网膜细胞）。 </li><li>离心细胞，收集适当（ 例如 ，3分钟，在405×g离心PC12细胞或在218×g离心RMC1细胞10分钟），除去上清，重悬在8ml适当的细胞培养介质。 </li><li>如果混合视网膜细胞将被使用，从通过酶消化（木瓜蛋白酶）P0-5新生大鼠幼仔按既定的方法19制备</li><li>计数存活的细胞不包括0.4％（重量/体积）的台盼蓝染料的数量，使用血球。 </li><li>调整细胞密度SUCh上的细胞将是约70-80％汇合在培养24或48小时后。 （对PC12细胞的接种密度为4.0×10 5个活细胞/ ml时，为RMC1细胞，它是2.5×10 5个活细胞/ ml和混合视网膜细胞为8×10 5个活细胞/ ml）中。加入细胞后，无论是透明的或黑色的96孔板（用于H 2 O 2或分别DCFH-DA分析），在100微升合适的生长培养基，使板坐在平坦表面上，在37℃，5 ％的CO 2（或其他条件都适合于特定的细胞类型）中至少24小时，以允许细胞附着。 </li><li>培养的细胞在适当的96孔托盘中的生长培养基，直到他们70-80％汇合（24小时为PC12和RMC1细胞，48小时混合视网膜细胞）。 </li></ol> 3.添加谷氨酸应激到细胞<ol><li>准备L-谷氨酸单钠盐水合物应激0-10MM的浓度适当满成长个培养基。 </li><li>从细胞中除去培养基，并用PBS轻轻（PC12）或HBSS（RMC1和混合视网膜细胞）洗涤细胞3次。后洗涤后，含谷氨酸介质添加到100微升的总体积/孔。 </li></ol>光处理的四首剂量<ol><li>立即在加入谷氨酸或选择的压力源，通过将制备的光递送装置，根据细胞暴露于光处理在所期望的波长和强度。一定要改变光束的使用的依赖于波长的中性密度滤光片的既定的组合的量子输出被施用。 注意:在当前的实验中，细胞暴露于光处理3分钟，由搁在96孔板上37℃热垫保持体温。根据需要治疗的时间可以变化。 <ol><li>放置了磨砂黑卡的细胞下方，以防止光反射到邻近井在96 WEL升盘指定的其他治疗参数。 </li></ol></li><li>如果治疗所需的时间较长的长度，添加的25mM Hepes缓冲液以维持PH值的细胞培养基或理想地，将装置和处理板的培养箱内用CO 2浓度调节到5％的CO 2，或条件是最优对于特定的细胞类型。 </li><li>以下的光治疗结束后，将细胞在一个水平面上，并培育在37℃和5％的CO 2（或其他条件，最适合于细胞类型）24小时。 注意:根据需要，如上所述R / NIR-LT的附加剂量可以施用，。 </li></ol>光治疗ROS和检测5.最终用量<ol><li>之前进行最后一轮的光处理，准备用于活性氧的检测试剂。制备的50mM柠檬酸缓冲液（pH6.0的），加入Triton X100和H 2 O 2的检测试剂的工作溶液（1:2:97的比例的10mM的H 2 O 2的检测试剂原液; 10 U / ml的辣根过氧化物酶; 50mM的柠檬酸钠（pH 6.0）中）。制备DCFH-DA在100μM的在适当培养基的最终浓度。 注意:DCFH-DA试剂PC12细胞在RPMI培养基，DCFH-DA试剂RMC1细胞是在DMEM介质。需要注意的是可商购的检测试剂具有差的敏感性，以特定的活性氧和试剂应仔细选择，以提供所需的单个应用程序中的信息。 </li><li>辖光治疗的细胞中，如在步骤4.1-4.1.2说明。确保在细胞被治疗的光的期望的能量密度/波长。 </li><li>紧接光处理，取出含有谷氨酸媒体和与适当的缓冲液洗两次（用于PC12细胞，PBS中被使用，对于RMC1细胞的HBSS时）。对细胞进行ROS测定如下: <ol><li> H 2 O 2 </suB&gt;试验:添加45微升的50mM柠檬酸盐缓冲液（pH6.0）和5μl的Triton X100到每个孔中。轻轻摇动细胞上轨道摇床进行30秒，在37℃和5％CO 2的15分钟。加入50微升的H 2 O 2的检测工作溶液孵育30分钟，在室温。使用读板器以530nm的的激发波长和480纳米的发射波长测量荧光。 </li><li> DCF测定:添加100微升的100μM的溶液DCFH-DA到每个孔中，并孵育30分钟，在37℃和5％的CO 2。除去含有100μM的DCFH-DA中的媒体，并与适当的缓冲液洗涤（如上所述）的两倍。加入90微升缓冲溶液和10微升的Triton X100到每个孔中，并在37℃下轻轻摇动在轨道摇床上分钟温育30秒前15。使用读板器以480纳米的激发波长和发射沃维勒测量的DCF荧光衍生ength 530nm处的。 </li><li>明示的ROS值相对于剩余孔中的细胞的蛋白质浓度，使用比色试剂盒根据制造商的说明书来定量蛋白浓度，参照标准曲线来计算mg蛋白质。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

我们已经成功地适应了精确和校准的光传输系统，以提供一种机制，用于对R / NIR-LT 在体外优化的研究。的R波长和强度的参数/ NIR-LT能够被准确有效地操纵使用这个系统。我们建立了光治疗的细胞并没有导致细胞死亡，尽管ROS没有减少在输送的波长和剂量，在测试的细胞类型。当前系统在670nm处（20.11W /米2）实现的最大强度在过量照射的外显率的先前公布的措施到大鼠脑，其中1.17瓦...

活性氧（ROS）的所需要的各种信号转导途径和细胞代谢的正常反应，包括那些神经保护1。然而，当内源性抗氧化机制无法控制ROS的产生，细胞可能会屈从于氧化应激2,3。损伤后的中枢神经系统，在ROS和氧化应激的存在相关联的增加被认为在损伤4,5的进展显着的作用。尽管用于衰减已评估氧 ​​化应激策略的广泛的数目，目前有用于以下神经外伤6衰减ROS产生和相关的氧化应激在临床使用中没有完全有效，临床相关抗氧化剂的策略。因此氧化应激的衰减仍是治疗介入7的一个重要目标。 后续的改进荷兰国际集团的R / NIR-LT已经报道了在广泛的伤害和疾病，包括减少心肌梗死面积，糖尿病期间肾和肝的并发症，视网膜变性，中枢神经系统损伤和中风8，或许通过减少氧化应激。特别是关于CNS损伤，670nm处光的功效的临床前研究已经显示在视网膜变性9-11，脊髓损伤12，神经元死亡13的模型良好的效果。临床试验已经进行了干性年龄相关性黄斑变性和目前正在进行对于中风14，但是这些试验的结果不出现希望的，或许是由于未能采用有效的治疗参数15。因此，R / NIR-LT并没有被广泛采用作为在神经外伤正常临床实践的一部分，尽管是一种简单的施用，非侵入性的和相对便宜的治疗方法。障碍的临床翻译，包括缺乏CL的早知道行动规范有效的治疗方案16,17和缺乏机制。关于光疗法目前的文献揭示了过多的变化治疗参数相对于照射源（LED或激光），波长（ 例如 ，630，670，780，810，830，880，904nm），照射总剂量（焦耳/单位面积），持续时间（曝光时间），定时（前或后损伤），治疗的频率和传递模式（脉冲或连续）8。在研究之间的治疗参数的变化，使比较困难，已经向有关的功效持怀疑态度16贡献。 因此，R / NIR-LT的优化显然需要，与细胞培养系统能够提供必要的比较的多个变量的高通量筛选的机制。然而也有一些市售的照明系统，可以提供足够的灵活性和控制わvelength和强度进行这样的优化实验。市售的LED器件通常是不能够提供多个波长或强度，从而导致研究者使用来自不同制造商，其可以不仅在强度变化的多个LED器件，而且光发射的波长的光谱。我们已通过使用宽带氙光源透过窄带干涉滤光片过滤，从而产生范围内的波长和光的能量密度，从而允许R / NIR-LT的参数密切，精确控制解决了这一问题。 要注意的是治疗的治疗剂量由光子与photoacceptor（发色团），其中在R / NIR-LT的情况下被假定为细胞色素C氧化酶（COX）18相互作用的数目定义是很重要的。输送光子能量随波长;意相等剂量的能量以不同的波长将是玉米珍贵的不同数量的光子的。因此，从装置发射的光被校准以发射相等数目的光子的每个所选择的波长进行测试。我们已开发了可用于递送的R / NIR-LT在一定范围的波长和强度，以细胞在体外的系统和演示来测量递送的R /对ROS产生NIR-LT在细胞经受影响的能力谷氨酸压力。

<table><tbody><tr><td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td><td>Cell Biolabs</td><td>STA-342</td><td></td></tr><tr><td>Amplex UltraRed Reagent</td><td>Molecular Probes</td><td>A36006</td><td></td></tr><tr><td>300 Watt Xenon Arc Lamp</td><td>Newport Corporation</td><td>6258</td><td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td></tr><tr><td>USB4000-FL Fluorescence Spectrometer</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>CC-3-UV Cosine Corrector for Emission Collection</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>200&amp;mu;m diameter quartz fibre optic</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>SpectraSuite Spectroscopy Platform</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>2300 EnSpire Multimode Plate Reader</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>9002-93-1</td><td>Acute toxicity, wear gloves when handling.</td></tr><tr><td>L-Glutamic acid monosodium salt hydrate</td><td>Sigma-Aldrich</td><td>142-47-2 (anhydrous)</td><td></td></tr><tr><td>Pheochromocytoma rat adrenal medulla (PC12) cells</td><td>American Type Culture Collection</td><td>CRL-2522</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI1640) Media</td><td>Gibco</td><td>11875-119</td><td></td></tr><tr><td>Fetal Bovine Serum, certified, heat inactivated, US origin</td><td>Gibco</td><td>10082-147</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>Horse Serum, New Zealand origin</td><td>Gibco</td><td>16050-122</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050-061</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>100 mM Sodium Pyruvate</td><td>Gibco</td><td>11360-070</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140-122</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>100X MEM Non-Essential Amino Acids Solution</td><td>Gibco</td><td>11140-050</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>Retinal Muller (rMC1) cells</td><td>University of California, San Diego</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11965-118</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Flasks</td><td>BD Biosciences</td><td>B4-BE-353136</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Sigma-Aldrich</td><td>25988-63-0</td><td>Aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14025-134</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Gibco</td><td>10010-049</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>Laminin Mouse Protein, Natural</td><td>Gibco</td><td>23017-015</td><td>Aliquot and store at -20 &amp;deg;C</td></tr><tr><td>1X Neurobasal Medium</td><td>Gibco</td><td>21103-049</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>165U Papain</td><td>Worthington</td><td></td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>W326305</td><td></td></tr><tr><td>Corning 96 well plates, clear bottom, black</td><td>Corning</td><td>CLS3603-48EA</td><td></td></tr><tr><td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td><td>Costar</td><td>07-200-103</td><td></td></tr><tr><td>Seesaw Rocker</td><td>Standard lab epuipment</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Standard lab epuipment</td><td></td><td></td></tr><tr><td>Neutral Density Filter Paper (0.3)</td><td>THORLABS</td><td></td><td></td></tr><tr><td>442 nm Bandpass Filter</td><td>THORLABS</td><td>FL441.6-10</td><td></td></tr><tr><td>550 nm Bandpass Filter</td><td>THORLABS</td><td>FB550-10</td><td></td></tr><tr><td>670 nm Bandpass Filter</td><td>THORLABS</td><td>FB670-10</td><td></td></tr><tr><td>810 nm Bandpass Filter</td><td>THORLABS</td><td>FB810-10e</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.1)</td><td>THORLABS</td><td>NE01B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.2)</td><td>THORLABS</td><td>NE02B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.3)</td><td>THORLABS</td><td>NE03B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.5)</td><td>THORLABS</td><td>NE05B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.6)</td><td>THORLABS</td><td>NE06B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (1.0)</td><td>THORLABS</td><td>NE10B</td><td></td></tr></tbody></table>

method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

红/近红外光疗法（R / NIR-LT），通过激光或发光二极管（LED）的递送，可能通过减少氧化应激改善功能和形态学效果的范围内的中枢神经系统损伤的体内 ，的。然而，在氧化应激的R / NIR-LT的作用已被证明取决于波长或辐射的强度而变化。在比较治疗参数的研究缺乏，由于缺乏能够提供多个波长或强度，适用于通过量高的体外优化研究商用设备。这个协议描述了用于递送光的技术在一定范围的波长和强度，以优化所需的一个给定的损伤模型治疗剂量。我们假设，提供光的方法，其中波长和强度参数可以很容易地被改变，可以促进确定的R / NIR-LT的最佳剂量的减少活性氧（ROS）的体外。 非相干氙光通过窄带干涉滤光片过滤，以提供不同的波长（440，550，670的中心波长和810纳米）和能量密度（8.5×10 -3至3.8×10 -1焦耳/ cm 2）的光的向培养的细胞中。从该装置的光输出被校准以发射治疗相关，等于量子剂量的光的每个波长。检测反应性物质在谷氨酸强调与光处理的细胞，用DCFH-DA和H 2 O 2的敏感的荧光染料。 我们成功地在一个范围内的生理学和治疗相关的波长和强度的光传递，以暴露于谷氨酸作为CNS损伤模型的培养细胞。而R / NIR-LT在当前研究中使用没有施加由培养的细胞产生的活性氧的效果的能量密度，轻的递送方法也适用于其他SYSTEMS包括分离线粒体或多种生理上相关的器官切片培养模型，并且可以用来评估的范围内的氧化代谢的结果的措施的效果。

使用中性密度滤光片，以照射细胞的范围内的能量密度包含先前证明是有益的体内 （0.3焦耳/厘米2）20 670nm处的光的剂量的光在670nm处的波长传送的输出进行校准。作为在光源增加前中性密度滤光片的数量，强度（W /米2）降低，从而允许更少的光传递到目标区域。表1给出了670nm处的光的从装有一个波长滤光器，光源产生的校正数据，并包括用于ND过滤器的数量和光的作为结?...

Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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Research

JoVE Journal

Biology

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

8.7K 次观看.  The University of Western Australia. Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

视频: Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

科研

癌症研究

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

生物化学

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

发育生物学

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 &#176;C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 &#956;M can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

使用硫代巴比妥酸反应物质测定法评估生物样品中的氧化应激

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

化学

GSH/GSSG 测量 – 用于抗氧化剂研究的改进 OPA 检测

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30&#181;M (R2=0.9932&#177;0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

作者聚焦：创新巯基定量和生物标志物检测用于氧化应激研究 

Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

免疫与感染

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法

摘要

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此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

使用硫代巴比妥酸反应物质测定法评估生物样品中的氧化应激

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在 vivo小鼠创面模型中活性氧的成像

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法

摘要

探索更多视频

此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

使用硫代巴比妥酸反应物质测定法评估生物样品中的氧化应激

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在 vivo小鼠创面模型中活性氧的成像

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽