Name: an in-house-built and light-emitting-diode-based photodynamic therapy device for enhancing verteporfin cytotoxicity in a 2d cell culture model
Uploaded: 2023-01-13T12:40:00
Description: Watch this Scientific Journal Video about An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model at JoVE.com

我们感谢亚瑟·恩里克·戈麦斯·德·奥利维拉和卢卡斯·朱利安·克鲁兹·戈麦斯对拍摄过程的帮助。该项目得到了巴西研究委员会（CNPq，资助号400953/2016-1-404286/2021-6）和南洋杉基金会-PPSUS 2020/2021（SUS2020131000003）的支持。这项研究也部分由巴西苏必利尔尼维尔的财政代码001资助。

注意：有关此协议中使用的所有材料、试剂和软件的详细信息，请参阅 材料表 。
1. 设备结构
<ol><li>锯切3毫米厚的中密度纤维板（MDF），以获得尺寸如图 1A所示的碎片。 注意： 使用矢量文件（补充文件 1）进行计算机数控 （CNC） 切割。</li>
	<li>构建两个具有以下尺寸（长 x 宽 x 高）的盒子：较大的盒子为 330 mm...

<ol>
	<li>Ferlay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+agency+for+research+on+cancer.">International agency for research on cancer.</a> Global Cancer Observatory: Cancer Today. 23 (7), 323-326 (2018).</li><li>Gottesman, M. M., Fojo, T., Bates, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+resistance+in+cancer:+role+of+Atp-dependent+transporters.">Multidrug resistance in cancer: role of Atp-dependent transporters.</a> Nature Reviews Cancer. 2 (1), 48-58 (2002).</li><li>Szakacs, G., Paterson, J. K., Ludwig, J. A., Boothe-Genthe, C., Gottesman, G. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+cancer:+what's+past+is+prologue.">Photodynamic therapy for cancer: what's past is prologue.</a> Photochemistry and Photobiology. 96 (3), 506-516 (2020).</li><li>Barr, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contrasting+mechanisms+of+colonic+collagen+damage+between+photodynamic+therapy+and+thermal+injury.">The contrasting mechanisms of colonic collagen damage between photodynamic therapy and thermal injury.</a> Photochem Photobiol. 46 (5), 795-800 (1987).</li><li>Algorri, J. F., Ochoa, M., Rold&#225;n-Varona, P., Rodr&#237;guez-Cobo, L., L&#243;pez-Higuera, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy:+A+compendium+of+latest+reviews.">Photodynamic therapy: A compendium of latest reviews.</a> Cancers. 13 (17), 4447 (2021).</li><li>Aniogo, E. C., Plackal, B., George, B. P. A., Abrahamse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+photodynamic+therapy+on+multidrug+resistant+breast+cancer.">The role of photodynamic therapy on multidrug resistant breast cancer.</a> Cancer Cell International. 19, 91 (2019).</li><li>Spring, B. Q., Rizvi, I., Xu, N., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+photodynamic+therapy+in+overcoming+cancer+drug+resistance.">The role of photodynamic therapy in overcoming cancer drug resistance.</a> Photochemical &amp; Photobiological Sciences. 14 (8), 1476-1491 (2015).</li><li>Dougherty, T. J., Grindey, G. B., Fiel, R., Weishaupt, K. R., Boyle, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoradiation+therapy.+II.+Cure+of+animal+tumors+with+hematoporphyrin+and+light.">Photoradiation therapy. II. Cure of animal tumors with hematoporphyrin and light.</a> Journal of the National Cancer Institute. 55 (1), 115-121 (1975).</li><li>Etcheverry, M. E., Pasquale, M. A., Garavaglia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+HeLa+cell+cultures+by+using+LED+or+laser+sources.">Photodynamic therapy of HeLa cell cultures by using LED or laser sources.</a> Journal of Photochemistry and Photobiology B: Biology. 160, 271-277 (2016).</li><li>Guo, Q., Dong, B., Nan, F., Guan, D., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Aminolevulinic+acid+photodynamic+therapy+in+human+cervical+cancer+via+the+activation+of+microRNA-143+and+suppression+of+the+Bcl-2/Bax+signaling+pathway.">5-Aminolevulinic acid photodynamic therapy in human cervical cancer via the activation of microRNA-143 and suppression of the Bcl-2/Bax signaling pathway.</a> Molecular Medicine Reports. 14 (1), 544-550 (2016).</li><li>Mroz, P., Yaroslavsky, A., Kharkwal, G. B., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+pathways+in+photodynamic+therapy+of+cancer.">Cell death pathways in photodynamic therapy of cancer.</a> Cancers. 3 (2), 2516-2539 (2011).</li><li>Mahalingam, S. M., Ordaz, J. D., Low, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+a+photosensitizer+to+the+mitochondrion+enhances+the+potency+of+photodynamic+therapy.">Targeting of a photosensitizer to the mitochondrion enhances the potency of photodynamic therapy.</a> ACS Omega. 3 (6), 6066-6074 (2018).</li><li>Granville, D. J., Levy, J. G., Hunt, D. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+treatment+with+benzoporphyrin+derivative+monoacid+ring+A+produces+protein+tyrosine+phosphorylation+events+and+DNA+fragmentation+in+murine+P815+cells.">Photodynamic treatment with benzoporphyrin derivative monoacid ring A produces protein tyrosine phosphorylation events and DNA fragmentation in murine P815 cells.</a> Photochemistry and Photobiology. 67 (3), 358-362 (1998).</li><li>Castano, A. P., Demidova, T. N., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+in+photodynamic+therapy:+part+two+-+cellular+signaling,+cell+metabolism+and+modes+of+cell+death.">Mechanisms in photodynamic therapy: part two - cellular signaling, cell metabolism and modes of cell death.</a> Photodiagnosis Photodynamic Therapy. 2 (1), 1-23 (2014).</li><li>Detty, M. R., Gibson, S. L., Wagner, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+clinical+and+preclinical+photosensitizers+for+use+in+photodynamic+therapy.">Current clinical and preclinical photosensitizers for use in photodynamic therapy.</a> Journal of Medicinal Chemistry. 47 (16), 3897-3915 (2004).</li><li>Allison, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy:+oncologic+horizons.">Photodynamic therapy: oncologic horizons.</a> Future Oncology. 10 (1), 123-142 (2014).</li><li>Chepurna, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+with+laser+scanning+mode+of+tumor+irradiation.">Photodynamic therapy with laser scanning mode of tumor irradiation.</a> Optical Fibers and Their Applications 2015. 9816, 323-326 (2015).</li><li>Huang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+progress+in+clinical+photodynamic+therapy.">A review of progress in clinical photodynamic therapy.</a> Technology in Cancer Research and Treatment. 4 (3), 283-293 (2005).</li><li>Chepurna, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED-based+portable+light+source+for+photodynamic+therapy.">LED-based portable light source for photodynamic therapy.</a> Optics in Health Care and Biomedical Optics. 11190, 109-115 (2019).</li><li>Hasson, O., Wishkerman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CultureLED:+A+3D+printer-based+LED+illumination+cultivation+system+for+multi-well+culture+plates.">CultureLED: A 3D printer-based LED illumination cultivation system for multi-well culture plates.</a> HardwareX. 12, 00323 (2022).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localised+light+delivery+on+melanoma+cells+using+optical+microneedles.">Localised light delivery on melanoma cells using optical microneedles.</a> Biomedical Optics Express. 13 (2), 1045-1060 (2022).</li><li>Erkiert-Polguj, A., Halbina, A., Polak-Pacholczyk, I., Rotsztejn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+diodes+in+photodynamic+therapy+in+non-melanoma+skin+cancers-own+observations+and+literature+review.">Light-emitting diodes in photodynamic therapy in non-melanoma skin cancers-own observations and literature review.</a> Journal of Cosmetic and Laser Therapy. 18 (2), 105-110 (2016).</li><li>Neupane, J., Ghimire, S., Shakya, S., Chaudhary, L., Shrivastava, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+light+emitting+diodes+in+the+photodynamic+therapy+of+rheumatoid+arthritis.">Effect of light emitting diodes in the photodynamic therapy of rheumatoid arthritis.</a> Photodiagnosis and Photodynamic Therapy. 7 (1), 44-49 (2010).</li><li>Lins, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+785-nm+laser+diode-based+system+for+standardization+of+cell+culture+irradiation.">A novel 785-nm laser diode-based system for standardization of cell culture irradiation.</a> Photomedicine and Laser Surgery. 31 (10), 466-473 (2013).</li><li>Hopkins, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+vitro+cell+irradiation+protocol+for+testing+photopharmaceuticals+and+the+effect+of+blue,+green,+and+red+light+on+human+cancer+cell+lines.">An In vitro cell irradiation protocol for testing photopharmaceuticals and the effect of blue, green, and red light on human cancer cell lines.</a> Photochemical and Photobiological Sciences. 15 (5), 644-653 (2016).</li><li>Zhang, K., Waguespack, M., Kercher, E. M., Spring, B. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+and+stable+LED+array+illumination+system+for+multiwell+plate+cell+culture+photodynamic+therapy+experiments.">An automated and stable LED array illumination system for multiwell plate cell culture photodynamic therapy experiments.</a> Research Square. , 1-18 (2022).</li><li>G&#225;lvez, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+evaluation+of+the+operational+characteristics+of+a+new+photodynamic+therapy+device.">Analysis and evaluation of the operational characteristics of a new photodynamic therapy device.</a> Photodiagnosis and Photodynamic Therapy. 37, 102719 (2022).</li><li>Bretin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+activity+of+new+human+colorectal+cancer.">Photodynamic therapy activity of new human colorectal cancer.</a> Cancers. 11 (10), 1474 (2019).</li><li>. T. SketchUp Available from: <a target="_blank" href="https://www.sketchup.com/">https://www.sketchup.com/</a> (2022)</li><li>LCDR PhotoDynamic Therapy (PDT) Equipment Repository. GitHub, Inc Available from: <a target="_blank" href="https://github.com/PhotoDynamicTherapy">https://github.com/PhotoDynamicTherapy</a> (2022)</li><li>W3C CSS Color Module Level 3. W3C, Inc Available from: <a target="_blank" href="https://www.w3.org/TR/css-color-3/#SRGB">https://www.w3.org/TR/css-color-3/#SRGB</a> (2022)</li></ol>

最终的PhotoACT设备使用市售的低成本组件建造起来很方便，总成本不到50美元。其他优点包括低维护需求，能够照射多种类型的培养板，每次测定同时使用多达四个单位，重量轻（2 kg）/尺寸（44 cm3），便于携带，准确且可重复的辐照（数据未显示），以及用户友好且简单的设置界面，无需连接到计算机或其他机器。
施工和运营协议的某些关键步骤在项目构思期间产生...

在最致命的非传染性疾病中，癌症是导致过早死亡的全球主要原因。2020年，它造成近1000万人死亡，约占全球六分之一的死亡1。此外，多重耐药性（MDR）现象构成了巨大的公共卫生威胁，因为批准的化疗方案未能达到该临床状况的缓解阶段2。癌细胞可以通过多种机制对化疗产生耐药性;然而，一些ATP结合盒（ABC）转运蛋白（ATP依赖性外排泵）的过表达被认为是肿瘤微环境中MDR发展的主要原因3。除耐多药外，其他癌症并发症，如复发和转移，也加强了对开发和改进治疗方法以克服这一肿瘤学挑战的迫切需求。
光的治疗利用已经实践了几个世纪4，光动力疗法（PDT）代表了临床认可的实体瘤治疗方法。PDT结合光敏剂（PS）的施用，然后进行光照射以产生活性氧（ROS），以在肿瘤细胞中发挥选择性细胞毒性。这种治疗方法优于传统方法，包括手术、放疗和化疗5;它是一种微创技术，在结缔组织中显示出较低的细胞毒性6。直接在肿瘤或其微环境中的光应用和PS积累确保了精确的靶向，因此，轻微的，不良的全身副作用7以及在同一部位重复治疗的可能性。此外，成本低于其他方法。由于其有希望的特性，PDT可以被认为是单一的适当选择，特别是在无法手术的肿瘤的情况下，或辅助癌症治疗7，并且代表了与化疗相关的MDR的替代方法8，9。
第一份报告显示使用PDT的高客观反应率于1975年在小鼠和大鼠模型10中描述。从那时起，使用PDT进行了研究，在2D细胞培养物中与人肿瘤细胞系在体内和体外均取得了积极的结果711，12。考虑到临床认可的PS的广泛适用性，无论其特定的积累途径和吸收峰的波长范围如何，一般过程如下：（i）PS摄取，（ii）肿瘤或其微环境中PS浓度的峰值，（iii）光应用，（iv）PS光相互作用，（v）PS激发态能量转移到组织底物或周围的氧分子， （vi） ROS产生，涉及单线态氧或超氧阴离子，（vii）肿瘤细胞死亡，本质上是坏死或凋亡（直接死亡），自噬（细胞保护机制），组织缺血（血管损伤），免疫调节或这些机制的重叠7。在这个最后阶段，特定细胞死亡途径的激活取决于许多因素，例如细胞特征，实验设计以及最重要的是PS细胞内定位和PDT相关的靶向损伤13。
维替泊芬是第二代PS，在挪威和中国被监管机构批准用于临床使用7。据报道，给药后，该前药在线粒体14 中部分积累并诱导细胞蛋白酪氨酸磷酸化和DNA片段化，导致肿瘤细胞凋亡15，16。孵育24小时后用于维特泊芬内化，建议使用690nm波长设置的PDT方案，以实现向相邻分子7，17的有效电磁辐射转移水平。
关于PDT的光源，经典的二极管激光系统通常价格昂贵，技术复杂，尺寸过大，因此不便携18，19。由于其单波长分布（也可以在基于LED的PDT设备中观察到），每个光敏剂应用对独立单元的需求使得二极管激光系统的使用更加复杂，经济上不可行20，21。因此，利用LED机械被认为是最有前途的替代方案，不仅可以解决成本22和维护问题，而且还要提供高功率输出和危害较小的23以及更宽的照明能力24，25，26，27。
尽管基于LED的设备可以为PDT实验28提供潜在的贡献，但大多数商业选择仍然存在缺点，例如缺乏便携性，高成本以及复杂的建筑项目和操作29。这项工作的主要目的是为 体外 PDT测定提供一种简单可靠的工具。本文介绍了 PhotoACT，这是一种内部构建的基于 LED 的 PDT 设备，价格低廉、用户友好且便于携带。作为概念验证，该装置被证明可以增强维替泊芬在2D细胞培养模型中的细胞毒性，因此可以用作PDT实验中的研究工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% Trypsin-EDTA (10x)</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td>Mammalian cell culture dissociation reagent</td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Flashforge</td>
			<td></td>
			<td>Finder model</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td></td>
			<td></td>
			<td>Non-sterile, polystyrene, and high-binding surface plates with flat bottom wells used for 2D cell culture</td>
		</tr>
		<tr>
			<td>Arduino</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brightness sensor</td>
			<td></td>
			<td></td>
			<td>TSL2561 model with 0.1-40.000+ lux detection levels and I2C interface</td>
		</tr>
		<tr>
			<td>Buttons</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buzzer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Flasks</td>
			<td></td>
			<td></td>
			<td>Sterile, polystyrene, rectangular bottom flask with Tissue Culture (TC)-treated surface, canted neck and vent cap (sizes)</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td></td>
			<td></td>
			<td>Sterile, polypropylene tubes with 15/50 mL capacity used for cell culture dilution at seeding step of the assay</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Controller board</td>
			<td></td>
			<td></td>
			<td>ESP32</td>
		</tr>
		<tr>
			<td>Design Software</td>
			<td>Trimble</td>
			<td></td>
			<td>SketchUp</td>
		</tr>
		<tr>
			<td>DMEM High Glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D4540-500ML</td>
			<td>Dimethyl sulfoxide, &#8805;99.5% (GC), suitable for plant cell culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco</td>
			<td>12657029</td>
			<td>FBS provides the best value by delivering consistency of cell growth over time and passages.</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15750060</td>
			<td>Water-soluble antibiotic drug originally purified from the fungus&#160;Micromonospora purpurea. Gentamicin acts by preventing cell culture contamination</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td>Neubauer patterned chamber used for cell counting at seeding step of the assay</td>
		</tr>
		<tr>
			<td>Inverted Laboratory Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DM IL LED</td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td>Cabin designed to protect the working environment from contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area. Used at several steps of cell cultivation and treatment procedures</td>
		</tr>
		<tr>
			<td>LCD display</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LED RGB WS2812</td>
			<td></td>
			<td></td>
			<td>5050 RGB SMD model with a built-in processor. Tape with 30 LEDs, 1 meter length and 9 watts</td>
		</tr>
		<tr>
			<td>MDF fiberboards</td>
			<td></td>
			<td></td>
			<td>3mm thickness medium-density fiberboards</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td></td>
			<td></td>
			<td>Sterile, polypropylene tubes with safety lid and 1.5/2.0 mL capacity. Convenient tools for manipulating small volumes at treatment step of the assay</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>ThermoFischer</td>
			<td></td>
			<td>Multiskan FC Microplate Photometer designed to detect a broad wavelength range of absorbance (340-850 nm). The equipment was used to evaluate cell viability after MTT incubation.</td>
		</tr>
		<tr>
			<td>MTT Reagent</td>
			<td>Invitrogen</td>
			<td>M6494</td>
			<td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Used for cell viability assays</td>
		</tr>
		<tr>
			<td>Operational System</td>
			<td>Real Time Engineers ltd.</td>
			<td></td>
			<td>FreeRTOS</td>
		</tr>
		<tr>
			<td>P10 micripipette</td>
			<td></td>
			<td></td>
			<td>Non-electronic, single-channel, 1-10 &#956;L capacity</td>
		</tr>
		<tr>
			<td>P1000 micropipette</td>
			<td></td>
			<td></td>
			<td>Non-electronic, single-channel, 10-1000 &#956;L capacity</td>
		</tr>
		<tr>
			<td>P200 micropipette</td>
			<td></td>
			<td></td>
			<td>Non-electronic, single-channel, 20-200 &#956;L capacity</td>
		</tr>
		<tr>
			<td>PDT Equipment</td>
			<td>LumaCare</td>
			<td></td>
			<td>Model LC-122</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline pH 7.4</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>Balanced salt formulation used for washing cells during cultivation and assay procedures</td>
		</tr>
		<tr>
			<td>Potentiometers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tips</td>
			<td></td>
			<td></td>
			<td>Non-sterile, universal fit, 10/200/1000 &#956;L maximum volumes</td>
		</tr>
		<tr>
			<td>Verteporfin</td>
			<td>Sigma-Aldrich</td>
			<td>SML0534-5MG</td>
			<td>Verteporfin, &#8805;94% (HPLC)</td>
		</tr>
	</tbody>
</table>

an in-house-built and light-emitting-diode-based photodynamic therapy device for enhancing verteporfin cytotoxicity in a 2d cell culture model

本文描述了一种新颖、简单且低成本的体 外 光动力疗法 （PDT） 测定设备，名为 PhotoACT。该器件使用一组传统的可编程发光二极管（LED）、液晶显示器（LCD）模块和连接到商用微控制器板的光传感器构建。原型的盒式结构由中密度纤维板（MDF）制成。内部隔室可同时分配四个细胞培养多孔微孔板。
作为概念验证，我们研究了光敏剂（PS）维特泊芬在二维（2D）培养中对HeLa细胞系的细胞毒性作用。用增加浓度的维替泊芬处理HeLa细胞24小时。弃去含药物的上清液培养基，用磷酸盐缓冲盐水（PBS）洗涤贴壁细胞，加入无药培养基。在这项研究中，使用红绿蓝（RGB）值255、255和255（平均通量为49.1±0.6J / cm2）检查维替泊芬对细胞的影响。24小时后，通过3-（4，5-二甲基-2-噻唑基）-2，5-二苯基四唑溴化物（MTT）测定评估细胞活力。
实验结果表明，用维替泊芬处理的细胞暴露于来自装置的光下，通过由活性氧（ROS）介导的机制 增强了 药物的细胞毒性作用。此外，通过将结果与商用PDT设备进行比较，验证了这项工作中描述的原型的使用。因此，这种基于LED的光动力疗法原型代表了PDT体 外 研究的良好替代方案。

最终的PDT设备名为PhotoACT，包括一个暗室，用于分配多达四个多孔微孔板，其上内表面配备了一组30个散射LED，编程为发射不同的可见光光谱（图3 和 补充文件6）。该设备使用两个相关的盒子构建：一个设计为PDT测定暗室的内部盒子，以及一个覆盖内部室并固定控制单元的外部盒子（图1B）。内部盒子被漆成黑色（图1D</strong...

Watch this Scientific Journal Video about An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model at JoVE.com

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

在这里，我们描述了一种新颖，简单且低成本的设备，可以使用二维HeLa细胞培养物和维特泊芬作为光敏剂成功进行 体外 光动力疗法（PDT）测定。

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...

光动力疗法（PDT）为癌症治疗提供了几个优点，其疗效取决于光源来激活光敏剂。尽管该领域取得了最新进展，但缺乏用于体外模型的昂贵且可重复的PTD设备。为了满足这一需求，这项工作描述了一种新颖，简单且低成本的设备，用于对细胞培养物进行PDT测定，称为PhotoAct。要开始施工，锯三毫米厚的中密度纤维板，MDF，以获得具有以下尺寸的碎片。构建两个具有以下尺寸的盒子。钻较大盒子的背面以安装桶形插孔连接器。还要在大箱子的顶部、小箱子的顶部和底部钻孔，为电缆提供通道。用黑色墨水涂刷所有内表面，以促进均匀的入射光。在较小盒子的上内表面并联连接三个 LED 胶带，每个胶带有 10 个 LED。此外，在较小盒子的底部内表面的中心安装亮度传感器。使用补充 3D 打印文件打印控制单元的结构。安装所有组件、电源按钮、电位器、时间启动触摸板、LED、亮度传感器、LCD、蜂鸣器和电源，以及安装在控制单元内部的 ESP-32 控制器板的部件。上传补充文件中提供的编程代码，并运行测试以检查所有连接是否正常工作。组装盒子并将它们固定在一起，以避免间隙，从而避免外部照明干扰和即时光损失。将安装的控制单元连接到原型顶部的钻孔区域。在以下尺寸上建造相同材料的前门，并用铰链和魔术贴胶带将其固定在外盒上，以确保腔室关闭和不间断分析。还要安装一个把手，以轻松准确地操纵前门。在原型底部安装四个橡胶脚垫，以确保操作过程中更加稳定。在Dulbecco的改良鹰培养基低葡萄糖中培养HeLa细胞系，含10%胎牛血清和1%庆大霉素。将培养瓶保持在5%的二氧化碳和37摄氏度。管理和检查细胞培养物，直到达到80%至90%的汇合度。通过接种过程启动细胞活力方案。用融合的HeLa细胞培养物从烧瓶中取出培养基。用磷酸盐缓冲盐水PBS洗涤烧瓶，并按照突出显示的细节用胰蛋白酶分离培养物。用血细胞计数器计数重悬细胞，并将它们接种到每孔浓度为20， 000个细胞的多孔微孔板中。为深色和浅色处理条件准备两个板，并将它们孵育24小时以附着细胞。为了继续用光敏剂处理，从两个板上去除培养基，并用100微升浓度增加的维替泊芬处理细胞。将细胞保持在治疗中24小时，以使维替泊芬内化。孵育后，取出处理，用PBS洗涤细胞并加入无药物培养基。用铝箔覆盖一个微板以保护其免受光照，并将其孵育 24 小时。该微孔板将为PDT结果的进一步分析提供对照数据。另一个微孔板将在PhotoAct的光暴露条件下使用。要操作设备，请将其插入插座并打开，按电源按钮。将多孔微孔板放在PDT室，并通过用侧面魔术贴胶带固定前门来关闭设备。要设置设备，请使用电位计调整光发射的 RGB 配置。按加/减触摸板调整时间配置并设置检测持续时间。检查显示屏上是否显示了有关测定的正确信息，并在必要时进行最终调整。按下起始触摸板开始测定。在实验开始时必须听到一声蜂鸣器。在实验过程中，可以在显示屏上观察到进度信息，例如辐照度和剩余时间。在PDT测定过程中不要打开前门或改变任何配置。在测定结束时，必须听到四声蜂鸣器，电子系统将关闭所有 LED。在显示屏上可以观察到完成的消息和实验过程中膨胀的最终能量。根据突出显示的公式计算能量最终值。盖上遭受光照的微孔板，然后进行24小时孵育。孵育期后，从两个平板中取出培养基，用PBS洗涤单层细胞，并加入MTT溶液。将两个板在黑暗和明亮的条件下孵育四个小时，以形成甲臜晶体。小心地取出MTT溶液，并用DMSO和乙醇溶液溶解紫色晶体。晶体完全溶解后，使用酶标仪在595纳米处进行吸光度测量。最终产品由一个暗室组成，其上内表面配备了一组30个散射发光二极管（LED），编程为发射不同的可见光光谱。由于内表面的低反射率和LED配置的均匀分布，建立了均匀的入射光。设置界面用户友好，稳定的实验条件可重复。作为概念验证，该装置用于增强维替泊芬在光照后2D HeLa细胞培养中的细胞毒性作用。如图所示，在光照条件下，GI50值为3.1微摩尔。和 13.8 微摩尔用于黑暗条件 因此，与条件相比，效率提高了四倍以上，证实了维替泊芬作为光敏剂的使用以及 PhotoAct 在 PDT 测定中的适用性。为了验证这项工作中描述的原型的使用，在相同的实验条件下使用了商用PDT设备，包括光敏剂，细胞和通量，并比较了结果。如图所示，两种装置均等地光活化维替泊芬，增强了细胞毒性作用。最后，使用DCFDA测定法通过流式细胞术证实，使用DCFDA测定法确认了光照暴露后由维替泊芬引发的ROS介导的细胞死亡。总之，该设备很容易用市售的低成本组件制造，总成本不到50。该设备的其他主要优点包括便携性、低维护需求、能够照射多种类型的培养板、每次测定同时使用多达四个单元、准确且可重复的照射、用户友好和简单的设置界面，不需要连接到计算机或其他机器。此外，还提供了决策流程图，以提供系统的问题解决方法，以查找和纠正操作过程中的问题或错误。这些发现允许将PhotoAct的益处扩展到科学研究，探索光敏剂的作用机制及其临床应用。

Research

JoVE Journal

Biochemistry

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

通过对Rho激酶活性的暂时抑制,增强人类诱导多能干细胞衍生心肌细胞的诱导

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

使用免疫增强技术的超高速西布洛特

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...