The authors would like to thank the staff of the Syracuse University Department of Physics workshop for their innovative assistance in matters relating to our system design.

1.清洗细胞超声发生器系统<ol><li>清洁喇叭和转炉用氯仿和棉花棉签之间的结合。施加光或矿物油到喇叭和转换器的螺纹以进一步除去的金属屑，生锈，或氧化堆积。 </li><li>用氯仿擦拭螺纹油后，从工会除去油和其他污染物。 </li></ol> 2.重新组装细胞超声发生器和设置系统<ol><li>重新装上喇叭的转换器。到正确的扭矩系统，通过拉动喇叭出杯（ 图2A）的底部取出组装转换器和喇叭从杯组件。放置槽扳手内邻近所述转换器（ 图2B）的顶部的三个小孔中的一个。然后，放置一个扭矩扳手装配有半英寸乌鸦的脚放在喇叭（ 图2B）的开槽部分。拧紧以sTANDARD顺时针方向，直到扭矩计的读数54.23Ñ。米（ 图2C），制造商推荐的16的转矩。适合收紧器和喇叭回放入杯中，并安装喇叭和转换器环形支架在竖直位置。设置系统实验脉冲给药前重新将转换器的电源。 </li><li>设置系统使用1秒超声处理的脉冲间1秒脉冲给药。不同的给药模型可以被用于其它实验条件。许多变量，例如水体积和喇叭直径将可能要求改变此剂量。使用上述细胞样品类型，体积和浓度，经验数据和观察已导致这个给药方案的制剂。 </li><li>该系统调整到所需的幅度。在这个协议中，使用33％和50％的幅度，以防止完整细胞破坏，这将使sonosensit的功效izers很难或不可能测量。 注:在本研究中的幅度进行比较。 </li></ol> 3.评估系统强度和功能<ol><li>持气穴计在1.5厘米，这是样品超声处理的水平。注意读数来评估，该系统是在一个预期的强度运行。 100-110瓦/厘米2的33％的振幅和115-125瓦/厘米2的50％的幅度之间使用当前系统和水位，所期望的。注意从仪表显示器，其表示气穴能量在直接瓦/厘米2的气穴计读数。采取这些读数在水的表面上，并确保没有气泡存在于米探针的面。 注:需要注意的是，这些程序是没有上限的访问喇叭上面做的是非常重要的。他们不必在每次超声处理后重复。相反，它们应该只清洁，重新组装，以及设置系统之后进行。 </LI&gt; <li>将水听器在含有以充分浸入水听传感器12.5毫升水样品瓶。暂停使用环形支架上的水听器。 </li><li>附加的水听到示波器的输入以评估波形特性。检查示波器有一个明确的正弦波，如异常波可以从制造商的规格改变系统的频率。两个空化仪和示波器得到频率阅读，但只有示波器将显示异常波是指示有故障系统或装配不当的。 </li></ol> 4.制备细胞和培养基的<ol><li>通过使用240毫升的Iscove氏改良的Dubecco氏培养基（IMDM）和50毫升胎牛血清制备介质。不解冻的胎牛血清中的热水浴，如快速的温度上升会导致在血清稳定性的折衷。结合240微升庆大霉素50毫克/毫升和5 ml青霉素/链霉素100倍在标准培养瓶中，并加入到培养基中。储存在4℃的培养基。 </li><li>种子U937细胞在〜4×10在使用制备的介质的25cm 2烧瓶4个细胞/ ml。评估细胞用于使用TC20细胞计数器浓度和活力，并通过将15-20微升的细胞和15-20微升台盼蓝的成微离心管中并混合这些物料台盼蓝排斥。 Pipette15-20微升该混合物成TC20采样滑动，并通过将滑入TC20细胞计数器开始计数。 </li><li>孵育细胞在37℃和5％的CO 2，并检查是否有细胞活力用台盼蓝排除和TC20细胞计数器。当细胞是在适当的浓度和/或已经用sonosensitizers为适当的时间范围内，转印3毫升从培养瓶中的细胞，以20毫升玻璃闪烁瓶中。在这个协议中，生长的U937细胞48小时，然后用0.5％，1治疗，或5mM的MeβCD30分钟以充分耗尽之前超声处理细胞的胆固醇。 </li></ol> 5.细胞超声<ol><li>脱气去离子，用真空和Buchner瓶蒸馏水。水转移到牛角杯，并填写在15毫米以上的喇叭的顶部水平。超声处理过程引起的水进一步脱气一段几分钟，并可能导致不一致在实验的过程中，如果系统事先不运行采样超声处理。因此，运行系统的采样超声〜前7分钟。 </li><li>附加含有细胞到保持装置中的闪烁瓶中。然后，将保持装置在杯角的顶部和设置为（使用滑动机构在水面）从喇叭的前15毫米的高度。 </li><li>将细胞通常超声处理超声三个1秒的脉冲，以1秒的每个这些脉冲之间的间隔中。为了这协议，使用上述脉冲给药声处理细胞在33％和50％的幅度。 </li></ol> 6.评估细胞损伤后超声<ol><li>评估使用台盼蓝和TC20细胞计数悬浮细胞的损伤和可行性。此外，分析使用Z2计数器样品。为Z2计数器分析，吸管100微升细胞悬浮液于20ml等渗盐水（200:1的比例）。在分析前，用等渗盐水冲洗Z2颗粒分析器的孔径至少两次。放置在计数器保持器向Z2样品，并启动计数之前提高该平台的孔。 </li><li>获得使用由制造商提供的软件的TC20和Z2计数器的数据。分析这些数据对细胞损伤，活力，并从声处理创造细胞碎片。 </li></ol> 7. XTT检测，以确定细胞活力和治疗U937和THP1细胞线粒体活性<ol><li> PRIOR键的XTT测定法，成长U937和THP1细胞相同的条件下进行。预处理细胞MeβCD的30分钟之前，超声处理相同的浓度范围内。像以前一样使用相同的超声参数除了电池使用的范围1-3一秒的脉冲现在超声间隔一秒除了开发出一系列的XTT套件，以评估损失。 注:很多这些步骤都是直接从XTT试剂盒说明书和采取只重复感兴趣实验室的便利。 </li><li>离心200×g离心10分钟收集细胞。先前所述在生长培养基中重悬细胞沉淀。重悬的细胞在约1×10 5个细胞/ ml。种子U937细胞以每孔100微升到平底96孔微量滴定板中一式三份，然后重复用于使用单独的96孔板THP1细胞。包括含有100微升单独作为完整的生长介质空白吸光度读数3对照孔。然后，孵育接种板24小时。 </li><li>除霜XTT试剂，并在37℃至使用前的活化试剂的两等份。漩涡等分轻轻直到透明解。加入0.1毫升活化试剂的至5.0的XTT试剂，其形成一96孔微量滴定板分析足以活化XTT溶液的毫升。重复该过程对于其他板块。 </li><li>添加50微升活化-XTT溶液的各孔中。返回板固定到细胞培养物的CO 2培养箱中培养2小时。摇动板轻轻以下潜伏期均匀分布橙色的阳性孔。测量含有细胞的测定孔和空白背景对照孔的吸光度用微滴定板读数器450-500纳米波长之间的波长。 </li><li>要么使用空白对照孔为零微量滴定板读数器，或从特定的结果中减去它们的平均值。再次测量所有的测定孔的吸光度在waveleng日之间630-690 nm和减去获得的450-500纳米值值。注:本次吸收测定有助于消除从分析结果非特异性读数。的XTT分析重复四次两种细胞系。 </li></ol>

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The authors declare that they have no competing financial interests.

为了达到最佳的效果，特别应小心仔细地定位样品并清洁变换器喇叭联合。在喇叭样品的放置是用于获得一致的细胞破坏，如从喇叭改变距离将改变的声焦点，并因此改变能量的样品暴露于重要。杯子喇叭内的声能可以使用气穴计找到最大的气穴的位置进行映射。此外，空化仪，连同示波器是用于确定声音强度的细胞被暴露于，以及波形的同质性是至关重要的。因此，这些仪器应该用于检测问题的系统，并且有助于确定哪些故障可能需要校正系统不稳定。 如前面提到的，低频系统可以在整个如果不是运行实验起到进一步脱气的水几分钟前样品超声处理。这个初始运行必须执行以产生在实验过程中相对脱气超声处理介质，从而一致的结果。此外，细胞不应该在或接近最大振幅评估sonosensitizers的功效时，作为增感的真实程度将是难以评估超声处理。在40 kHz的系统上使用33％的幅度是一个理想的环境，因为它产生显着的伤害，但提供sonosensitizers足够的空间来展示其功效，其表现与MeβCD对U937和THP1细胞（ 图7）。这些数据也证实，MeβCD敏感性多个白血病线以低频超声以剂量依赖的方式。 已经出现了大量的实验以较高频率为0.75兆赫的范围内进行到8 MHz表示膜内的空化气泡的证据通过超声处理17-19的产生。然而，questioNS仍然在问候超声诱导的细胞裂解18的确切机制。我们已经利用低频超声波15，这种现象由其他实验室20，证明所示的链路的流化细胞骨架之间和增加的声波灵敏度21。此外，我们已经发现，微丝-破坏剂如细胞松弛素B增强在多个白血病超声波灵敏度行，但不hHSCs或白细胞22，提示抑制肌动蛋白聚合的可能特别感兴趣的一个sonosensitizing机制。我们还观察到，长春新碱，微管破坏剂，抑制微管蛋白聚合23，24，显着地增加不同白血病类型的体外超声波灵敏度包括急性骨髓性白血病，慢性骨髓性白血病和急性淋巴性白血病。相比之下，稳定细胞骨架成分骨架定向剂（紫杉醇的ðjasplakinolide）出现，使细胞抵抗超声，通过细胞裂解22的低利率反映。总之，这些数据支持流化的肿瘤细胞的细胞骨架组分的假设确实是在增加的SDT 25的功效的重要因素。本研究还表明，胆固醇耗竭可能是另一种方法，通过它，以进一步增强肿瘤细胞的超声波的灵敏度，如MeβCD处理U937细胞被显着地增感至40千赫的超声波。 而我们的超声处理的协议已经证明，在体外显着的抗肿瘤活性，目前的方法被限制在培养和小脊椎动物模型，其能够适合于用于超声处理的小瓶一起工作。我们已经表明，斑马鱼可以使用脉冲低频超声（20千赫）被安全地进行超声处理，并且它们的耐受化疗剂是定量媲美剂量由鼠模型26的耐受性，这表明荷瘤斑马鱼，可以使用在初步调查，以评估这些协议的体内抗肿瘤活性。然而，施用化疗剂之前，哺乳动物模型的超声处理已经报道在MHz范围为1，并且这样的协议可以容易地扩展到包括低频超声波，以及胆固醇消耗的和细胞骨架的定向剂。 这种形式的SDT的潜在的临床应用可以涉及体外血液超声处理，其中抗肿瘤剂是静脉内给药（ⅳ）之前，血液被移除用于超声处理25。此方法除去由人体解剖学构成潜在声屏障，并可能破坏白血病原始细胞，以及转移从实体瘤的有效方法。它也有可能是胆固醇消耗的和细胞骨架的定向剂可能在那些已经被检查，在诊所中，企图改善这个治疗方法的功效的HIFU协议被使用。 在本研究中所描述的方法能够评估潜在sonosensitizers的值，并且还系统细化可以提高此用途。然而，也有很多使用这种超声波不可同日而语，包括电源质量，声灶和转换器之间的个体差异时要考虑的变量。因此，未来的研究将集中在可视化的声波，并了解他们对结果的影响。 SDT已显示增强细胞裂解的体外和可能被证明是如果有更多的在哺乳动物模型中的体内数据采集临床可行。实验研究的恶性细胞的涉及多个代理和超声其他潜在的可利用的特性，以及各种组合方式在我们的实验室继续。

超声是指具有一个频率大于人的听觉能力的上限（约20千赫兹）1任何振荡声压力波。低频超声在20-60千赫范围已被用于在实验室中作为产生的乳剂，制备蜂窝样品核酸提取，用于组织破坏的手段，以及用于各种其它的测试。低频超声的效用也已经延伸到工业环境中进行焊接，清洗各种材料，并在材料的加工。市售超声波发生器进来频率范围为18-60 kHz和全面的​​从瓦W. 100-1,200 虽然超声早已在临床上用于诊断成像时，它已被最近才应用于作为治疗方法。超声≥1兆赫能够安全地破坏泌尿系结石（肾结石）和胆道结石（结石日E在患者胆囊或肝脏），以减轻症状2,3。这种方法被称为体外冲击波碎石术（ESWL）现已广泛应用于临床（超过一百万的患者每年治疗ESWL在美国就有4），并提供了一个模式由以非侵入分手结石最小的附带损伤通过使用外部施加的，有重点，高强度的声波脉冲2-4。 由于独特的直接的剪切力，以及由高强度超声波产生空化气泡，这些方法已经被检查在癌症治疗的去势抗性前列腺癌和胰腺癌中被称为高强度的方法治疗聚焦超声（HIFU ）5-8。的方式非常相似的体外冲击波碎石，HIFU使用多个超声波束并把它们聚焦在选定的重点领域，以产生温度为60℃或HIG她通过使用声能的，诱导凝固性坏死，在靶组织5。尽管热消融的其他方式当前存在（射频消融和微波消融术），HIFU提供了一个独特的优势，因为它是唯一的非侵入性的方式温热5这些方法。 HIFU已取得不同的结果在门诊，目前仅在临床试验中可用8-11。然而，有限的成功它已经实现，并且非常有前途的 ，从临床前哺乳动物模型获取的体内数据证明超声波在癌症治疗的潜力。 在努力提高HIFU，研究人员试图超声具有适当的抗肿瘤剂结合生成sonochemotherapy的一种形式。声动力治疗（SDT）是一种有前途的新的治疗方式已经证明令人印象深刻的抗肿瘤活性在体外和<em&gt;体内研究1。它已经显示，超声优先损害基于这样的细胞和那些正常的组织学1,5之间的尺寸差是恶性细胞。 SDT包含称为sonosensitizers增加通过超声对肿瘤细胞施加优先损害的程度专门代理。而SDT的治疗应用已经预先检查，用超声系统通常使用较高频率的超声波（≥1兆赫），以及低千赫频率的超声波的作用还没有得到充分的探讨。超声的较低频率往往在产生惯性空化，这种现象将导致细胞的破坏更精通由于微泡的快速塌陷，诱导理化损伤12-14。在MHz和低千赫超声之间惯性空化的产生这种差别已被归因于一个事实，即低波频率使微泡š更多时间纠正扩散膨胀半周期在以下压缩半周期12成长，从而产生更猛烈的塌陷。 我们以前曾表明，U937人单核细胞白血病细胞对低频超声（23.5千赫）敏感，且这种敏感性可通过抗肿瘤剂的扰乱细胞骨架15的应用来显着增加。此外，我们已经证明，细胞被优先地基于大小破损，具有表现出较高的超声波灵敏度较大的单元。此外，在可比较的小区大小正常人类造血干细胞（hHSCs）和白细胞得多超声处理比其肿瘤对口15更耐，姑且表明低频超声可以用于优先地破坏恶性细胞的正常组织的存在。 为了进一步检验独特的道具低频超声用于潜在治疗用途ERTIES，我们已经开发清洁和稳定程序增加我们当前超声处理系统中的一个的功效和可靠性，布兰森模型SLPE 150瓦，40千赫细胞破碎，配备有20mm的喇叭装到7.62cm的杯子。此外，我们已经能够利用空化仪和示波器与水听器的40千赫的范围内确定准确的样品空化能量，以及一致的波形和幅度。通过提炼和系统化我们的协议，我们已经能够建立一致性在我们的实验超声处理，使我们能够定量比较不同的组织发生谱系的肿瘤细胞和正常细胞的敏感性声音。我们对40千赫的系统协议，提出了广泛的细节，以便感兴趣的实验室能够执行对比实验，并评估我们的通过引起的抗肿瘤效果的研究结果低频超声。此外，我们研究甲基β环糊精的剂量依赖效应（MeβCD; 图1），胆固醇耗竭剂，在增加的U937的超声波灵敏度和THP1人单核细胞白血病细胞。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium w/ NaHCO3 &#38; 25mM Hepes&#160;</td>
			<td>Life Technologies</td>
			<td>12440079</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B Solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A2942&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x Solution&#160;</td>
			<td>Life Technologies</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12105</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson SLPe 40kHz Cell Disruptor with 3&#34; (25mm) Cuphorn</td>
			<td>Branson Ultrasonics</td>
			<td>101-063-726</td>
			<td>sonication device</td>
		</tr>
		<tr>
			<td>Brisk Heat SDC Benchtop Digital temperature Controler w/ 1000mL Beaker Heater</td>
			<td>Brisk Heat</td>
			<td>SDCJF1A-GBH1000-1</td>
			<td>heater used for temperature control</td>
		</tr>
		<tr>
			<td>Beckman-Coulter Z2 Cell Sizer with AccuComp&#174; Software</td>
			<td>Beckman-Coulter</td>
			<td>6605700</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td>145-0102</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin 50mg/mL</td>
			<td>Sigma-Aldrich</td>
			<td>G1397&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL &#38; 25mL Vented Culture Flasks</td>
			<td>Fisher Scientific</td>
			<td>353082</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza L-Glutamine 200mM 0.85% NaCl</td>
			<td>Lonza</td>
			<td>17-605C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Seal-Rite 1.5 mL Microcentrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman-Coulter Accuvette ST 25mL Vials and caps</td>
			<td>Beckman-Coulter</td>
			<td>&#160;A35473</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuJet Pro Auto Pipet&#160;</td>
			<td>BrandTech Scientific</td>
			<td>26330</td>
			<td></td>
		</tr>
		<tr>
			<td>USA Scientific 10mL Disposable Serological Pipets</td>
			<td>USA Scientific</td>
			<td>1071-0810</td>
			<td></td>
		</tr>
		<tr>
			<td>Tip One 100uL and 1000uL Filter Tips</td>
			<td>USA Scientific</td>
			<td>1120-1840, 1126-7810</td>
			<td></td>
		</tr>
		<tr>
			<td>100uL Micropipette&#160;</td>
			<td>Wheaton</td>
			<td>851164</td>
			<td></td>
		</tr>
		<tr>
			<td>1000uL Micropipette</td>
			<td>Wheaton</td>
			<td>851168</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad Dual Chamber Counting Slides</td>
			<td>Bio-Rad</td>
			<td>145-0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Scientific Dual chamber water jacketed Incubator</td>
			<td>Forma Scientific</td>
			<td>3131</td>
			<td></td>
		</tr>
		<tr>
			<td>Tektronix&#160; DPO 2002B Digital Phosphor Oscilloscope</td>
			<td>Tektronix</td>
			<td>DPO2002B</td>
			<td>used to measure the ultrasonic waveform</td>
		</tr>
		<tr>
			<td>PPB MegaSonics Model PB-500 Ultrasonic Energy Meter</td>
			<td>PPB Megasonics</td>
			<td>PB-500&#160;</td>
			<td>used to assess the sound intensity in W/cm2</td>
		</tr>
		<tr>
			<td>Teledyne RESON TC4013-1 Hydrophone</td>
			<td>Teledyne</td>
			<td>TC4013-1&#160;</td>
			<td>connects to the oscilloscope&#160;</td>
		</tr>
		<tr>
			<td>Wheaton 250mL Flasks</td>
			<td>Sigma-Aldrich</td>
			<td>Z364827</td>
			<td></td>
		</tr>
		<tr>
			<td>20mL Glass Scintillation Vials&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>Z190527</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman-Coulter Isotonic Saline Solution&#160;</td>
			<td>Beckman-Coulter</td>
			<td>N/A</td>
			<td>diluent for Z2 counter</td>
		</tr>
		<tr>
			<td>Chloroform 99%</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof Anhydrous&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>N/A</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>XTT Cell Proliferation Assay Kit</td>
			<td>ATCC</td>
			<td>30-1011K</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Microplate Reader</td>
			<td>Cole-Palmer</td>
			<td>&#160;EW-13055-54</td>
			<td></td>
		</tr>
		<tr>
			<td>U937 Human Monocytic Leukemia Cells</td>
			<td>ATCC</td>
			<td>CRL&#173;1593.2</td>
			<td></td>
		</tr>
		<tr>
			<td>THP1 Human Monocytic Leukemia Cells</td>
			<td>ATCC</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and quantitative analysis of pulsed low frequency ultrasound to determine the sonic sensitivity of untreated and treated neoplastic cells

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In addition, this method can be used in combination with specialized agents known as sonosensitizers to increase the extent of preferential damage exerted by ultrasound against neoplastic cells, an approach referred to as sonodynamic therapy (SDT). The methodology for generating and applying low frequency ultrasound in a preclinical in vitro setting is presented to demonstrate that reproducible cell destruction can be attained in order to examine and compare the effects of sonication on neoplastic and normal cells. This offers a means by which to reliably sonicate neoplastic cells at a level of consistency required for preclinical therapeutic assessment. In addition, the effects of cholesterol-depleting and cytoskeletal-directed agents on potentiating ultrasonic sensitivity in neoplastic cells are discussed in order to elaborate on mechanisms of action conducive to sonochemotherapeutic approaches.

系统的稳定性是最重要的获得可重复的和可靠的结果。的54.23ñ。米适当的扭矩规格取得的换能器和超声波发生器2的适当的连接，与波形的一致性是通过利用示波器和水听器（ 图3A）的评估。 图3B描述的样品瓶内测量，以及图3C评估杯内号角稳定。正如预期的那样，振幅略有减少由于一些能量被吸收的玻璃。然而，该波形被未扰动或任一面板B或C，这是必要的可靠递送声能与样品中扭曲。没有出现明显的奇异正弦波波形表示传感器出现故障或安装不当。这被表示在图3D中表示的多个异常波，明确不存在一个正弦波和频率读取无牛逼预期与正常工作的系统。用于测量所产生的气穴能量（ 图4）的气穴计发现声音强度是100-110瓦/厘米2，在33％的幅度，和125-130瓦/厘米2，在50％的幅度。 一旦40 kHz系统被适当校准和组装在其全部（ 图5），可靠的细胞破坏物容易获得，如通过TC20和Z2计数器（ 图6）都确定。正如预期的，更广泛的细胞损伤中观察到超声处理在50％，而不是在33％振幅的细胞群。然而，33％的振幅是作为一个起点，以检测给药剂的潜在的声波的敏化影响，因为它产生明显的损伤是有用的，但留有待检测的伤害时施加试剂以评估超声波灵敏度的增强作用。这表现在图7A中 ，在那里MeβCD对增效U937细胞的超声波灵敏度剂量依赖性效果呈现。虽然非超声MeβCD处理的细胞在存活非常类似于非超声处理，未经处理的细胞中，存活率后三个脉冲的1秒40千赫超声施加显着下降。此外，在超声处理后的降低存活率似乎已经剂量依赖性，如5毫MeβCD产生细​​胞计数的最大降幅，随后为1mM，然后0.5mM的MeβCD。 观察在被具有变化在超声处理之前浓度MeβCD的处理，所评估的XTT试剂盒（ 图7B）都U937和THP1细胞对细胞活力和线粒体活性类似的效果。虽然THP1细胞似乎是略微更声波性比U937细胞，人类白血病线被损坏以剂量依赖的方式。较高浓度的MeβCD和超声PROD的多个脉冲uced XTT的最低还原成可溶，颜色鲜艳的橙色衍生物为U937和THP1细胞，相应降低细胞活力和线粒体活性。 <img alt="图1" src="/files/ftp_upload/53060/53060fig1.jpg" /> 甲基β环糊精图1.分子结构。（A）的甲基β环糊精（MeβCD）的完整结构。 （B）的 MeβCD是七个重复单元与一个R基团是H或CH 3的聚合物。分子式:C 56ħ98Ø35。分子量= 1331.36。 <img alt="图2" src="/files/ftp_upload/53060/53060fig2.jpg" /> 图2.正确torqueing的40千赫的超声波系统。（A）喇叭和转换器从杯中取出PRI或torqueing。 （B）放置槽扳手和扭矩扳手，在各自的岗位上。再度将喇叭和转换器到杯前（C）的应用的扭矩54.23ñ。米以顺时针方向。 <img alt="图3" src="/files/ftp_upload/53060/53060fig3.jpg" /> 图3.使用示波器到超声系统的波形的特征 。 （A）与水听器的示波器可以用于评估振幅和波形的一致性。 （B）中的记录采取与杯喇叭正上方1.5厘米，设定在33％的幅度。 （C）图为波形是从收购时的水听器放在20毫升玻璃闪烁瓶中之内阅读。将小瓶定位在1.5厘米喇叭上面并再次设定在33％的幅度。 （D）的示波器截图ØFA系统异常的频率，将有望与故障系统或喇叭变频器工会不正当耦合。 <a href="https://www.jove.com/files/ftp_upload/53060/53060fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53060/53060fig4.jpg" /> 图4.使用的气穴计来表征超声系统的声音强度。这里看到的气穴计用于表示空化能量在瓦/厘米2。这是用于评估相对于所述样本的能量的一致性非常重要的工具。 <img alt="图5" src="/files/ftp_upload/53060/53060fig5.jpg" /> 40千赫的超声波系统图5.关键组件。超声波系统显示完整的角杯（ <stro纳克&gt; A）中，加热装置（B）中，转换器（C），以及样品定位机构（D）。这种设计可实现样品（E）的喇叭（F）以上容易定位。 <img alt="图6" src="/files/ftp_upload/53060/53060fig6.jpg" /> 图6.影响振幅对使用TC20和Z2计数器U937细胞的超声裂解不同的水平 。将细胞超声处理用33％振幅超声三个1秒脉冲40千赫细胞dismembrator（100-110瓦/厘米2）或50％的幅度（125-130瓦/厘米2），以在各间1秒间隔这些脉冲。 （ 一 ）从Z2抗衡的软件的屏幕截图展示了超声对U937细胞的作用。 （ 二）从TC20计数器数据进行编译的曲线图展示了结果的重现性秒。这些数据包括总的和活细胞计数，以及台盼蓝评估细胞生存力。酒吧反映平均值（SEM）为4个独立的细胞群的标准差。 <a href="https://www.jove.com/files/ftp_upload/53060/53060fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53060/53060fig7.jpg" /> 上增效U937和THP1细胞的超声波灵敏度甲基β环糊精图7.影响。（A）的 U937细胞与不同MeβCD的浓度为30分钟被超声处理用40千赫超声（105瓦/厘米之前处理2 ）用三个1秒间隔的脉冲1秒间隔。细胞群体被分为≥12微米≥17微米。 （B）中的U937或THP1细胞用不同concent再次处理MeβCD的30分钟被超声使用1-3脉冲1秒超声40 kHz系统之前口粮间隔1秒间隔。细胞活力和线粒体活性进行了评估与XTT套件。将100μl细胞每孔接种到平底96孔微量滴定板。该板之前加入XTT溶液温育24小时。然后将细胞温育另外2小时的波长读取之前。酒吧反映SEM的4个独立的细胞群。 <a href="https://www.jove.com/files/ftp_upload/53060/53060fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells at JoVE.c

Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells

Selective damage of human leukemia cells can be achieved through a novel approach of applying low frequency ultrasound both with and without chemotherapeutic pretreatment of leukemic and normal hematopoietic cells.

代脉冲式低频超声定量分析，以确定未处理的声波的敏感性和治疗肿瘤细胞

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In ...

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Research

JoVE Journal

Medicine

11.1K 次观看.  Syracuse University. Selective damage of human leukemia cells can be achieved through a novel approach of applying low frequency ultrasound both with and without chemotherapeutic pretreatment of leukemic and normal hematopoietic cells. 

视频: Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

科研

医学

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

在疾病的染色质蛋白质组的定量分析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

在家兔模型宫内生长受限胎儿超声心动图和多普勒超声脉冲波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

小鼠膈肌功能诊断超声成像

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型:胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...