这项工作由中国国家自然科学基金 (81300810 号)、上海市青年医生培训计划 (20141057 号) 和中国国家自然科学基金 (81671982、81271791和 81571955) 资助。我们感谢 LetPub (www.letpub.com) 在编写本手稿期间提供语言援助。

1. 生物膜形成
<ol><li>
96井微型生物膜的形成
	<ol>
<li>检索金黄色葡萄球菌应变 USA300 和3生物膜形成的临床菌株 (C1-C3), 存储在-80 摄氏度。 注: 临床菌株形成生物膜的能力由前述的微量滴定板检测方法确定(15)。</li>
		<li>在5毫升的胰蛋白胨大豆汤中接种细菌, 并在一个孵化器中培养, 一夜之间以37摄氏度的温度抖动到固定的阶段。</li>
		<li>将隔夜细菌培养在25摄氏度的 4000 x g 上离心, 然后丢弃上清。并用重悬磷酸盐缓冲盐水 (PBS) 中的颗粒, 最终浓度为 2.0 x10 9 CFU/毫升。 注: 通过测量光学密度并进一步确定了细菌浓度, 并通过平板计数16, 揭示了 1 OD600的悬浮液中含有 1.5 x 108 CFU/毫升。</li>
		<li>将细菌悬浮液稀释到含有0.5% 葡萄糖的 1:200 (1.0 x 107 CFU/毫升) 中。接种200µL 细菌悬浮液进入细胞培养处理过的聚苯乙烯 96-井微板块。</li>
		<li>在含氧良好的环境下, 将微板块静态地孵化为24小时37摄氏度。 注: 成熟生物膜形成的潜伏期可能因不同菌株而异;这应在 PDT 实验15之前确定。</li>
		<li>在井中丢弃介质, 用 PBS 轻轻冲洗微板块井三次, 然后丢弃上清。 注意: 该步骤应非常温和地进行, 以避免扰乱形成的生物膜。</li>
	</ol>
</li>
	<li>
碟形中的生物膜形成
	<ol>
<li>将金黄色葡萄球菌菌株接种到5毫升的 USA300 中, 并在一个孵化室中培养, 在37摄氏度一夜之间摇晃到固定相。</li>
		<li>将隔夜细菌培养在25摄氏度的 4000 x g 上离心, 然后丢弃上清。将 PBS 中的颗粒并用重悬到最终浓度为 2.0 x10 9 CFU/毫升。然后, 将细菌悬浮液稀释到含有0.5% 葡萄糖的 1:200 (1.0 x 107 CFU/毫升) 中。将2毫升细菌悬浮液接种到35毫米光学质量的玻璃底部细胞培养皿中, 并以37摄氏度静态孵育24小时。 注: 通过测量光学密度来估计细菌的浓度。</li>
		<li>用吸管吸出培养基, 然后用 PBS 轻轻地冲洗盘子中的生物膜三次, 然后小心地丢弃上清液。 注意: 避免接触到盘子底部的吸管尖端。步骤2.2 应在这一步骤后立即执行, 以防止干燥的形成生物膜。</li>
	</ol>
</li>
</ol>2. 光辐照
<ol><li>在4摄氏度冰箱里存储 5-阿拉巴马。实验前, 用 PBS 稀释 5-阿拉-10 毫米。 注: 5-阿拉氨酸溶液应在实验前进行新的准备。</li>
	<li>在实验组, 添加200µL 10 毫米的微板块或2毫升到养殖皿的每一个井。用铝箔盖住盘子/盘子, 孵化1小时。然后, 用发光二极管 (LED) 照射板/盘, 亮度为100兆瓦/厘米2 , 为 1 h, 在 2 @ 633 nm10的主要波长处达到光剂量 360 J/厘米17 。 注: 为了让光能有效地和均匀地传递到所有水井/盘子中的生物膜, 将光源的峰值与6.0 厘米的井/盘的距离固定, 并将实验区限制在中心辐照区 (10 厘米 x 8cm). 为了确保结果是可重现的, 实验应在相同的室温下进行。 在 led 照射步骤中, 为了避免直接接触到其他光源 (如日光、房间照明或灯光), led 在将盘子/盘子移到辐照区前打开, 光线足够亮, 足以完成操作。</li>
	<li>设置控制组 (在实验中设置了三个控制组)。<ol>
<li>对于第一个控制组 (阿拉巴马 LED), 添加200µL 的 PBS, 每井微板块或2毫升的文化菜。用铝箔盖住盘子/盘子, 孵化2小时。</li>
		<li>对于第二个控制组 (阿拉巴马 + LED), 添加200µL 10 毫米的微板块或2毫升到养殖皿的每一个井。用铝箔盖住盘子/盘子, 孵化2小时。</li>
		<li>对于第三个控制组 (阿拉巴马 LED +), 增加200µL 的 PBS 微板块或2毫升的文化菜。用铝箔盖住盘子/盘子, 孵化1小时。然后将该板暴露在 LED 上, 其 360 J/厘米2光照射波长为 633 @ 10 nm17。 注: 在 LED 照射步骤中, 避免直接接触到其他光源, 如日光、房间照明或灯光。</li>
	</ol>
</li>
</ol>3. PDT 治疗效果的测定
注: 为了确认对金黄色葡萄球菌生物膜的影响, 通过 CFU 计数和活性染色法对具有或不带 pdt 的细胞的生存能力进行了评价。
<ol><li>
剩余存活菌细胞的测定
	<ol>
<li>治疗后, 丢弃在水井中的介质和冲洗与 PBS 三倍的水井, 以消除所有非黏附细胞的实验和对照组。 注意: 这一步应该非常温和地进行。</li>
		<li>用吸管尖端从水井中彻底刮去附着的细菌细胞, 并收集锥形管中的细胞。</li>
		<li>离心机的细菌悬浮在 4000 x g 10 分钟4摄氏度, 然后丢弃上清。</li>
		<li>并用重悬1毫升0.25% 胰酶酶在 PBS 中的细菌和孵育37°c 1.5 小时。</li>
		<li>离心机在 4000 x g 10 分钟;丢弃上清, 然后在 PBS 的200µL 中并用重悬颗粒。</li>
		<li>用 PBS 制作1:10 系列稀释细胞溶液;然后, 将每个系列稀释样品的5µL 添加到胰蛋白胨大豆琼脂 (TSA) 板上。孵育37°c 的 TSA 板16小时;然后, (肉眼) 计数细菌菌落数 (CFU/毫升)。</li>
	</ol>
</li>
	<li>
CLSM 对金黄色葡萄球菌生物膜的观察
	<ol>
<li>光照射后, 用 PBS 将培养基中的生物膜洗净三次。 注意: 这一步应该非常温和地进行。</li>
		<li>添加1毫升的1µM 绿色荧光核和染色体染色, 可渗透到原核细胞膜 (例如, SYTO9) 和1毫升1µM 碘碘 (PI), 以染色生物膜和死细胞。</li>
		<li>观察可行细胞 (绿色荧光, 前/em 485 nm/530 nm) 和死细胞 (红色荧光, 前/em 485 nm/630nm) 下 CLSM 与 63X 1.4-钠油浸泡物镜。</li>
		<li>使用显微软件生成图像。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riddle+of+biofilm+resistance.">Riddle of biofilm resistance.</a> Antimicrob Agents Chemother. 45 (4), 999-1007 (2001).</li><li>Rabin, 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=Biofilm+formation+mechanisms+and+targets+for+developing+antibiofilm+agents.">Biofilm formation mechanisms and targets for developing antibiofilm agents.</a> Future Med Chem. 7 (4), 493-512 (2015).</li><li>Mah, T. F., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+biofilm+resistance+to+antimicrobial+agents.">Mechanisms of biofilm resistance to antimicrobial agents.</a> Trends Microbiol. 9 (1), 34-39 (2001).</li><li>Sharma, M., 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=Toluidine+blue-mediated+photodynamic+effects+on+staphylococcal+biofilms.">Toluidine blue-mediated photodynamic effects on staphylococcal biofilms.</a> Antimicrob Agents Chemother. 52 (1), 299-305 (2008).</li><li>Rosa, L. P., da Silva, F. C., Nader, S. A., Meira, G. A., Viana, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effectiveness+of+antimicrobial+photodynamic+therapy+(APDT)+using+a+660+nm+laser+and+malachite+green+dye+in+Staphylococcus+aureus+biofilms+arranged+on+compact+and+cancellous+bone+specimens.">In vitro effectiveness of antimicrobial photodynamic therapy (APDT) using a 660 nm laser and malachite green dye in Staphylococcus aureus biofilms arranged on compact and cancellous bone specimens.</a> Lasers Med Sci. 29 (6), 1959-1965 (2014).</li><li>Rosa, L. P., Silva, F. C., Nader, S. A., Meira, G. A., Viana, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+antimicrobial+photodynamic+therapy+using+a+660+nm+laser+and+methyline+blue+dye+for+inactivating+Staphylococcus+aureus+biofilms+in+compact+and+cancellous+bones:+An+in+vitro+study.">Effectiveness of antimicrobial photodynamic therapy using a 660 nm laser and methyline blue dye for inactivating Staphylococcus aureus biofilms in compact and cancellous bones: An in vitro study.</a> Photodiagnosis Photodyn Ther. 12 (2), 276-281 (2015).</li><li>Mai, B., 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+antibacterial+effect+of+sinoporphyrin+sodium+photodynamic+therapy+on+Staphylococcus+aureus+planktonic+and+biofilm+cultures.">The antibacterial effect of sinoporphyrin sodium photodynamic therapy on Staphylococcus aureus planktonic and biofilm cultures.</a> Lasers Surg Med. 48 (4), 400-408 (2016).</li><li>Gandara, L., Mamone, L., Bohm, G. C., Buzzola, F., Casas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+photodynamic+inactivation+of+Staphylococcus+aureus+biofilms+by+disruptive+strategies.">Enhancement of photodynamic inactivation of Staphylococcus aureus biofilms by disruptive strategies.</a> Lasers Med Sci. 32 (8), 1757-1767 (2017).</li><li>Baltazar, L. M., 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=Antimicrobial+photodynamic+therapy:+an+effective+alternative+approach+to+control+fungal+infections.">Antimicrobial photodynamic therapy: an effective alternative approach to control fungal infections.</a> Front Microbiol. 6, 202 (2015).</li><li>Fernandes, T., Bhavsar, C., Sawarkar, S., D'Souza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+novel+approaches+for+control+of+dental+biofilm.">Current and novel approaches for control of dental biofilm.</a> Int J Pharm. 536 (1), 199-210 (2017).</li><li>De Sordi, 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=Development+of+Photodynamic+Antimicrobial+Chemotherapy+(PACT)+for+Clostridium+difficile.">Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile.</a> PLoS One. 10 (8), e0135039 (2015).</li><li>Harris, F., Pierpoint, L. <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+based+on+5-aminolevulinic+acid+and+its+use+as+an+antimicrobial+agent.">Photodynamic therapy based on 5-aminolevulinic acid and its use as an antimicrobial agent.</a> Med Res Rev. 32 (6), 1292-1327 (2012).</li><li>Donnelly, R. F., McCarron, P. A., Tunney, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+photodynamic+therapy.">Antifungal photodynamic therapy.</a> Microbiol Res. 163 (1), 1-12 (2008).</li><li>Shi, H., Li, J., Zhang, H., Zhang, J., Sun, H. <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+5-aminolevulinic+acid+photodynamic+therapy+on+Candida+albicans+biofilms:+An+in+vitro+study.">Effect of 5-aminolevulinic acid photodynamic therapy on Candida albicans biofilms: An in vitro study.</a> Photodiagnosis Photodyn Ther. 15, 40-45 (2016).</li><li>Zhang, Q. Z., 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=5-aminolevulinic+acid-mediated+photodynamic+therapy+and+its+strain-dependent+combined+effect+with+antibiotics+on+Staphylococcus+aureus+biofilm.">5-aminolevulinic acid-mediated photodynamic therapy and its strain-dependent combined effect with antibiotics on Staphylococcus aureus biofilm.</a> PLoS One. 12 (3), 0174627 (2017).</li><li>Chang, Y. 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=Rapid+single+cell+detection+of+Staphylococcus+aureus+by+aptamer-conjugated+gold+nanoparticles.">Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles.</a> Sci Rep. 3, 1863 (2013).</li><li>Barra, F., 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+and+Antibiotic+Therapy+in+Combination+to+Fight+Biofilms+and+Resistant+Surface+Bacterial+Infections.">Photodynamic and Antibiotic Therapy in Combination to Fight Biofilms and Resistant Surface Bacterial Infections.</a> Int J Mol Sci. 16 (9), 20417-20430 (2015).</li><li>St Denis, T. G., 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=All+you+need+is+light:+antimicrobial+photoinactivation+as+an+evolving+and+emerging+discovery+strategy+against+infectious+disease.">All you need is light: antimicrobial photoinactivation as an evolving and emerging discovery strategy against infectious disease.</a> Virulence. 2 (6), 509-520 (2011).</li><li>O'Neill, J. F., Hope, C. K., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+bacteria+in+multi-species+biofilms+can+be+killed+by+red+light+in+the+presence+of+toluidine+blue.">Oral bacteria in multi-species biofilms can be killed by red light in the presence of toluidine blue.</a> Lasers Surg Med. 31 (2), 86-90 (2002).</li><li>Li, 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=Effects+of+5-aminolevulinic+acid-mediated+photodynamic+therapy+on+antibiotic-resistant+staphylococcal+biofilm:+an+in+vitro+study.">Effects of 5-aminolevulinic acid-mediated photodynamic therapy on antibiotic-resistant staphylococcal biofilm: an in vitro study.</a> J Surg Res. 184 (2), 1013-1021 (2013).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+from+the+natural+environment+to+infectious+diseases.">Bacterial biofilms: from the natural environment to infectious diseases.</a> Nat Rev Microbiol. 2 (2), 95-108 (2004).</li><li>Elias, S., Banin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-species+biofilms:+living+with+friendly+neighbors.">Multi-species biofilms: living with friendly neighbors.</a> FEMS Microbiol Rev. 36 (5), 990-1004 (2012).</li><li>Wu, 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=Design+and+Proof+of+Programmed+5-Aminolevulinic+Acid+Prodrug+Nanocarriers+for+Targeted+Photodynamic+Cancer+Therapy.">Design and Proof of Programmed 5-Aminolevulinic Acid Prodrug Nanocarriers for Targeted Photodynamic Cancer Therapy.</a> ACS Appl Mater Interfaces. 9 (17), 14596-14605 (2017).</li></ol>

作者没有什么可透露的

PDT 一直是一个良好的研究治疗癌症治疗, 因为它是发明100年前的18。在过去的十年中, PDT 已被应用作为一种抗菌策略, 并显示了对某些耐药性致病菌的有效性12。与浮游状态相比, 细菌生物膜似乎对抗生素治疗的抗药性更有3, 而对生物膜的影响还没有得到充分的研究。
本文介绍了一种体外PDT 系统, 并对该模型对金?...

金黄色葡萄球菌是一种重要的革兰氏阳性病原体, 殖民了人类寄主的皮肤和粘膜。它形成生物膜的能力被认为是其发病机制的一个重要方面1。细菌生物膜是一种植入自产基质中的细菌群落, 由胞外高分子物质组成, 包括多糖、DNA 和蛋白质。这种基质在细菌感染的持续性中起着重要作用, 对人体免疫系统和目前的抗微生物治疗有很大的抵抗力,2。尽管抗生素对生物膜的影响有限, 但抗生素仍然是生物膜感染的主要治疗方法。先前已经表明, 生物膜中的细胞比它们的浮游相对物3更耐抗生素 10-1000 倍。因此, 需要采取替代战略来克服这一问题。
光动力疗法是一种治疗细菌感染的替代疗法, 它利用适当波长的光线激活光敏。这导致了活性氧种类 (ROS) 的生产, 这是致命的靶细胞通过扰乱细胞壁, 失活酶, 并损害 DNA4。这种多目标特性使得细菌难以对 PDT 治疗产生抗药性。
在以前的报告5、6中, 研究了 PDT 对细菌和真菌生物膜的抗菌作用, 其中包括甲苯胺蓝、孔雀石绿、亚甲基蓝、氯 e6 和卟啉等多种光敏. 7,8,9,10,11,12,13。5-阿拉, 药的实际光敏剂, PpIX, 其特点是其小分子量和快速清除12,14。这些优势给了阿拉 PDT 的主要潜力作为治疗应用。虽然对浮游细菌的影响已经研究了许多组12, 但对细菌生物膜的抗菌作用尚未阐明。同时, 比较以往研究的结果是很难的。其中一个原因是不同的协议被各种组使用。因此, 本协议描述了一种基于我们以前的工作15的一种用于 PDT 系统的体外模型。通过 CFU 计算和 CLSM 活性染色证实了该模型的有效性。

<table>
	<tbody>
		<tr>
			<td>Tryptone Soya Broth (TSB)</td>
			<td>OXOID</td>
			<td>CM0129B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone Soya Agar (TSA)</td>
			<td>OXOID</td>
			<td>CM0131</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTO9</td>
			<td>Thermo Fisher Scientific</td>
			<td>L7012</td>
			<td>The LIVE/DEAD BacLight Bacterial Viability Kits&#160;</td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>L7012</td>
			<td>The LIVE/DEAD BacLight Bacterial Viability Kits&#160;</td>
		</tr>
		<tr>
			<td>Pancreatin</td>
			<td>Sigma-Aldrich</td>
			<td>P3292</td>
			<td></td>
		</tr>
		<tr>
			<td>5-aminolevulinic acid (ALA)</td>
			<td>Fudan Zhangjiang Bio-Pharm</td>
			<td>3.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus strain USA300</td>
			<td>/</td>
			<td>/</td>
			<td>The source of USA 300 references &#8220;Tenover FC, Goering RV. J Antimicrob Chemother. 2009 Sep; 64(3):441-6&#8221;.</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus clinical strains (C1-C3)</td>
			<td>/</td>
			<td>/</td>
			<td>All clinical strains were isolated from patients with chronic rhinosinusitis in the Department of Otorhinolaryngology-Head and Neck Surgery, Eye and ENT Hospital of Fudan University [Zhang QZ, Zhao KQ, Wu Y, et al. PLoS One. 2017 Mar; 12(3): e0174627].</td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Corning Inc</td>
			<td>3599</td>
			<td>Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile</td>
		</tr>
		<tr>
			<td>Fluorodish</td>
			<td>NEST Biotechnology</td>
			<td>801001</td>
			<td>Glass bottom, Non-pyrogenic</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5417</td>
			<td>Eppendorf</td>
			<td>Z365998 |&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>SHKE4000</td>
			<td>MaxQ 4000 Benchtop Orbital Shakers</td>
		</tr>
		<tr>
			<td>Light emitting diode (LED)</td>
			<td>Wuhan Yage Optic and Electronic Technique CO</td>
			<td>LED-IB</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP8 confocal laser-scanning microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica LAS AF software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMARIS software</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro model to study the effect of 5-aminolevulinic acid-mediated photodynamic therapy on staphylococcus aureus biofilm

金黄色葡萄球菌(金黄色葡萄球菌) 是一种常见的人类病原体, 引起化脓性和系统性感染。金黄色葡萄球菌感染很难根除, 这不仅是由于抗药性菌株的出现, 而且是它形成生物膜的能力。近年来, 光动力疗法 (PDT) 已被指出是控制生物膜感染的潜在治疗方法之一。然而, 需要进一步的研究, 以提高我们对其对细菌生物膜的影响的认识, 以及潜在的机制。这篇手稿描述了一个体外模型的 PDT 与 5-氨基乙酰丙酸 (5-阿拉), 一个前兆的实际光敏剂, 原卟啉 IX (PpIX)。简单地, 成熟的金黄色葡萄球菌生物膜与阿拉巴马一起孵化, 然后暴露在光中。随后, 通过共聚焦激光扫描显微镜 (CLSM) 计算菌落形成单元 (CFUs), 并通过活性荧光染色可视化方法对金黄色葡萄球菌生物膜的抗菌作用进行了量化。具有代表性的结果表明, 对金黄色葡萄球菌生物膜具有较强的抗菌作用。该协议很简单, 可用于开发一种体外模型, 用于研究用 ALA PDT 治疗金黄色葡萄球菌生物膜的方法。在未来的研究中, 也可以借鉴其他光敏对不同菌株的光动力分析, 并进行最小的调整。

在 USA300 和三株临床菌株中, 与对照组 (以阿拉巴马为首的、以阿拉为主导的 led 和阿拉巴马为首的 +) 相比, 生物膜中细菌的生存能力下降了 (图 1)。
为证实 CFU 测定的结果, 观察其对金黄色葡萄球菌生物膜的抗菌作用, USA300 生物膜通过 CLSM 与活性染色进行可视化. 存活和死亡细胞分别染色绿色?...

Watch this Scientific Journal Video about An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm at JoVE.com

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

本手稿描述了一种研究 5-氨基乙酰丙酸介导光动力疗法 (ALA)对金黄色葡萄球菌生物膜的抗菌作用的协议。该协议可用于开发一种体外模型来研究细菌生物膜与 PDT 在未来的治疗。

用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

an-vitro-model-to-study-effect-5-aminolevulinic-acid-mediated

Research

JoVE Journal

Biology

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

7.8K 次观看.  Fudan University. 本手稿描述了一种研究 5-氨基乙酰丙酸介导光动力疗法 (ALA)对金黄色葡萄球菌生物膜的抗菌作用的协议。该协议可用于开发一种体外模型来研究细菌生物膜与 PDT 在未来的治疗。

视频: An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

传统美国宇航局后肢卸小鼠模型的一个交替的方法

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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

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 central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

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 ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

研究蛋白质和微RNA调制对脂肪细胞功能的影响的脂肪细胞培养模型

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

收获和分解:生物膜方法研究中被忽视的步骤

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the ...