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 border="0" cellpadding="0" cellspacing="0">
	<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&#956;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&#176;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&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;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&#176;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&#176;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&#176;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&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 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&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;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&#176;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&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;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>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;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 ...

method-for-assessment-effects-range-wavelengths-intensities-rednear

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

8.6K 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

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

斑马鱼的响应行为的检测，以测量光强的变化

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

科研

生物学

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

糖原生化滴定<em&gt;在体外</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

测量氧化应激性<em&gt;秀丽隐杆线虫</em&gt;在96孔微量滴定板

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

评估细菌素对小鼠肠道微生物的影响的方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...