作者要感谢Michael Solga和其他流式细胞术核心工作人员提供流式细胞术培训和服务。作者还要感谢Shiva Sai Krishna Dasa，Dustin K. Bauknight，Melissa A. Marshall，James C. Garmey，Chantel McSkimming，Aditi Upadhye和Prasad Srikakulapu在脂质体制备（SSKD，DKB），组织收获（MAM，JCG）以及流式细胞术染色和样品采集（AU，PS，CM）方面的帮助。这项工作得到了阿斯利康，R01HL 136098，R01HL 141123和R01HL 148109，AHA 16PRE30770007和T32 HL007284资助的支持。

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作者没有什么可透露的。

在这里，我们描述了一个由三部分组成的方案，用于（i）制备用荧光脂质染料标记并加载有抗糖尿病化合物tesaglitazar的脂质体，（ii）通过眶后注射将脂质体施用于小鼠，以及（iii）收获，处理和染色组织以通过流式细胞术检测细胞水平的脂质体摄取。该协议回顾了大约150μm脂质体的制备和脂肪，血液和脾脏摄取的评估。脂质体制备是可扩展的，主要在室温下进行，并利用反相蒸发来最大化药物负载和有机溶剂的去除。使用该协议，纯化的脂质体样品中可以达到高达2 mg / mL的tesaglitazar浓度。制备的脂质体可以在4°C的HEPES缓冲液中储存一年以上。根据我们的经验，它们证明了平均粒径的最小变化。在用10 kDa离心过滤器超滤分离脂质体与外部药物后，通过分光光度法证明了低于10%的药物含量损失。
在脂质体制备过程中，需要考虑一些关键步骤和因素。首先，协议步骤的顺序很重要，必须遵守。其次，上样替格列塔扎时所用溶液的pH值必须保持在7.4，以最大限度地提高溶解度和有效上样量。第三，设备和过滤器的正确组装确保每个步骤的输出具有适当的尺寸和纯度。例如，如果 100 纳米和 200 纳米滤光片组装不当，则可能会导致一批异质性和尺寸不合适的脂质体。第四，需要在载药前完全去除醋酸钙，以最大限度地将替格列塔唑转移到脂质体中。要测试乙酸钙的完全去除，请使用高速沉降去除脂质体，然后测量非脂质体溶液中的乙酸钙水平。第五，重要的是在每一步称重并记录添加到脂质体制剂中的所有材料的质量。这确保了可以计算出适当的浓度并保持所需的材料比例。最后，如果技术执行不当，则可能存在不希望的异质性水平。使用DLS和其他方法（如电子显微镜）彻底检查此参数非常重要。要提高均匀性，请考虑调整所选过滤器尺寸或堆叠两个过滤器。
此外，在完全实施此方案之前，计划和优化流式细胞术的对照和抗体组合至关重要（表1， 表3）。应检测抗体，以确保使用适当的浓度进行染色，并且荧光团之间的重叠最小。脂质体制备过程中所用染料的激发和发射也必须考虑在组合规划中。在我们的结果中，我们使用了DiD，它与异藻蓝蛋白（APC）和AlexaFluor 647等荧光团具有相似的激发和发射。因此，我们没有在抗体组合中选择与这些荧光团偶联的抗体。此外，同种型对照不包括在该协议中。这是因为为该方案选择的抗体是经过充分验证的市售抗体。但是，如果有兴趣使用以前未优化的抗体，请考虑在进行完整实验之前对感兴趣的组织进行针对同种型对照的抗体测试。
虽然该协议演示了如何从小鼠治疗后提取和处理血液，脾脏，腹股沟脂肪和附睾脂肪组织，但这种通用方法可以应用于其他组织。根据感兴趣的组织，可能需要改变以下组织的处理和消化方案:肺21，肝22，腹膜腔3，骨髓3，23，脑24。
这种方法的一个重要局限性是，每只动物只能在一个时间点评估摄取。因此，将该协议与其他非侵入性成像技术相结合或相应地计划以确保有足够的资源进行评估可能是有利的。细胞摄取和细胞周转的时间是需要考虑的重要因素:脂质体将在前24小时内在全身循环，并且取决于吸收脂质体的细胞的寿命或它们对摄取的反应，可能会发生细胞死亡或进一步吞噬作用。我们之前的研究表明，DiD+ 种群在不同时间点的种群特征发生了变化3。因此，评估较早的时间点或与目的机制生物学最相关的时间点的摄取非常重要。此外，虽然可以使用该协议对整个组织中的细胞摄取进行定量，但流式细胞术不能揭示组织定位。将这种方法与组织学方法相结合有助于解决这一限制。
通常，该协议补充了现有方法，例如组织学和全身荧光成像。随着流式细胞术工具和方法的不断进步，为越来越多的特异性细胞群开发更大的组合将成为可能。我们建议在上述方法之外使用该协议，因为这将改善细胞摄取的评估，并提供验证流式细胞术观察到的结果的机会。例如，是否应该发现脂肪组织中的大部分颗粒被流式细胞术的巨噬细胞吸收。可以保存，固定，切片和染色相同脂肪组织的额外等分试样的免疫荧光以获取巨噬细胞标志物，以验证细胞类型确实吸收脂质体。这种方法应该增加纳米颗粒生物分布测定的严谨性:验证细胞特异性靶向，量化细胞摄取，识别脱靶摄取，并希望提供信息以产生观察到的治疗结果的机制假设。该协议也可以适用于使用不同脂质体的未来研究，研究其他组织中的摄取，以及在肥胖和代谢障碍或任何其他疾病的情况下测试新化合物，其中纳米颗粒递送是一种可行的治疗选择。

随着对开发利用纳米颗粒药物递送的疗法的兴趣不断增长，制备和评估颗粒分布和摄取的方法必须继续推进，扩展，并为研究界所用1，2。该协议旨在评估在用装有tesaglitazar（一种过氧化物酶体增殖物激活受体（PPAR）-α/γ激动剂3，4）的DiD标记脂质体处理后在体内吸收脂质体的确切细胞类型。在这些研究中，我们能够评估脂质体tesaglitazar治疗直接影响哪些细胞类型，靶向部分的功效，并生成假设来解释我们观察到的治疗结果。此外，在多种细胞类型中建立的生物学功能表明吞噬细胞如巨噬细胞、树突状细胞和肝脏特异性库普弗细胞占据了大部分脂质体5，6，7。利用该协议，我们已经证明非经典吞噬细胞也可以吸收脂质体3，4。
该协议提供了一种优化的方法，用于溶解tesaglitazar，通过反相蒸发制备脂质体，并使用醋酸钙作为远程药物加载的引诱剂。所提供的方法可供许多实验室使用，并且缺乏难以获得的材料和需要高温的步骤。该方案产生大小的脂质体，最适合增加体内循环8。此外，正如Su等人所总结的那样，迄今为止，已经对评估体内脂质体分布和组织摄取的方法进行了深入研究和测试9。正电子发射断层扫描（PET），磁共振成像（MRI）和荧光分子断层扫描（FMT）方法用于量化组织特异性生物分布和摄取9，10，11。虽然这些方法已经过优化以最大限度地提高体内检测率，但它们仍然缺乏以细胞分辨率量化体内脂质体摄取的能力。这里介绍的方案旨在通过使用流式细胞术来满足这一需求。最后，对于该协议，细胞摄取缩小到包括脂肪组织在内的一些组织。越来越多的文献调查了在肥胖，代谢障碍和炎症的情况下使用纳米颗粒提供治疗的潜力12，13，14，15，16，17。因此，我们认为与处理和分析脂肪组织的有效方法共享协议很重要 - 脂肪组织是在这些病理中起重要作用的组织之一。

<table>
	<tbody>
		<tr>
			<td>1-mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10-mL syringe</td>
			<td>BD</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>25-gauge needle, sterile for retro-orbital injection</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>27-gauge needle, sterile for retro-orbital injection</td>
			<td>BD</td>
			<td>305620</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse B220 BV421</td>
			<td>Biolegend</td>
			<td>103251</td>
			<td>Clone RA3-6B2</td>
		</tr>
		<tr>
			<td>Anti-mouse CD115 PE</td>
			<td>eBioscience</td>
			<td>12-1152-82</td>
			<td>Clone AFS98</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b PerCP Cy5.5 antibody</td>
			<td>BD Biosciences</td>
			<td>550993</td>
			<td>Clone M1/70</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c APC ef780 antibody</td>
			<td>eBioscience</td>
			<td>47-0114-82</td>
			<td>Clone N418</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 PE CF594</td>
			<td>BD Biosciences</td>
			<td>562291</td>
			<td>Clone 1D3</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 FITC antibody</td>
			<td>BD Biosciences</td>
			<td>553061</td>
			<td>Clone 145-2C11</td>
		</tr>
		<tr>
			<td>Anti-mouse CD31 BV605</td>
			<td>Biolegend</td>
			<td>102427</td>
			<td>Clone 390</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 PerCP</td>
			<td>BD Biosciences</td>
			<td>557235</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80 PE Cy7</td>
			<td>Biolegend</td>
			<td>123114</td>
			<td>Clone BM8</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Gemini Bio-products</td>
			<td>700-107P</td>
			<td></td>
		</tr>
		<tr>
			<td>Desalting spin-column</td>
			<td>ThermoFisher</td>
			<td>89889, 89890</td>
			<td>Zeba spin column</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic Light Scattering, Nicomp 370</td>
			<td>Particle Sizing System, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIX &#38; PERM Cell Permeabilization Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>GAS004</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze sponges</td>
			<td>Dermacea</td>
			<td>441211</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>3393-1MU</td>
			<td></td>
		</tr>
		<tr>
			<td>Liposome extruder</td>
			<td>Millipore Sigma</td>
			<td>Z373400</td>
			<td>LiposoFast</td>
		</tr>
		<tr>
			<td>Live/Dead Aqua</td>
			<td>ThermoFisher Scientific</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanosight</td>
			<td>Malvern Instruments Ltd</td>
			<td>NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic lubricant</td>
			<td>Optixcare</td>
			<td></td>
			<td>20g/70 oz Sterile</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% w/v aq. soln., methanol free</td>
			<td>Alfa Aesar</td>
			<td>433689L</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene vial for mincing</td>
			<td>Wheaton</td>
			<td>986701</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Buchi</td>
			<td>Re111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Misonix</td>
			<td>XL2020</td>
			<td></td>
		</tr>
		<tr>
			<td>T/Pump Heat therapy pump and pad</td>
			<td>Gaymer Industries</td>
			<td>TP-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tesaglitazar</td>
			<td>Tocris</td>
			<td>3965</td>
			<td></td>
		</tr>
		<tr>
			<td>Track-etched polycarbonate membranes</td>
			<td>Thomas Scientific</td>
			<td>1141Z**</td>
			<td>Whatman, Nuclepore Polycarbonate hydrophilic membranes</td>
		</tr>
		<tr>
			<td>ZetaSizer/DLS-ELS system</td>
			<td>Malvern Instruments Ltd</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation, administration, and assessment of in vivo tissue-specific cellular uptake of fluorescent dye-labeled liposomes

人们对使用脂质体在体内递送化合物的兴趣日益浓厚，特别是对于靶向治疗方法。根据脂质体配方，脂质体可以优先被体内不同的细胞类型吸收。这可能会影响治疗颗粒的疗效，因为不同疾病的进展是组织和细胞类型特异性的。在该协议中，我们提出了一种使用DSPC，胆固醇和PEG-2000 DSPE以及脂质染料DiD作为荧光标记合成和荧光标记脂质体的方法。该协议还提供了一种在体内递送脂质体和使用流式细胞术评估脂质体的细胞特异性摄取的方法。这种方法可用于确定吸收脂质体的细胞类型，并量化脂质体摄取在细胞类型和组织中的分布和比例。虽然本协议中未提及，但额外的测定（例如细胞仪上的免疫荧光和单细胞荧光成像）将加强所做的任何发现或结论，因为它们允许评估细胞内染色。方案可能还需要根据感兴趣的组织进行调整。

脂质体生产
这里发表的结果与我们之前发表的工作3，4，20中的结果相似。利用此处介绍的方案，我们期望产生大小约为150-150-160 nm的脂质体。DLS 显示平均脂质体直径为 163.2 nm，zeta 电位为 -19.2 mV（图 1A）。低温电子显微镜（cryo-EM）成像显示环状脂质体（图1B），DLS图显示与平均直径的标准偏差相对较小（图1C）。
脂质体结合阳性需要 PBS 处理的对照
我们小组先前使用该协议的研究调查了体内给药一周后脂肪SVF，脾脏和血液中与脂质体结合的细胞亚群3，4。使用PBS处理的小鼠，用脂质体处理小鼠样品上使用的相同抗体组合对腹膜腔和脾脏细胞进行染色。治疗一周后收获组织（图2A）。来自PBS处理的小鼠的样品用作DiD FMO，用于创建阳性DiD门（图2B，C）。可以使用DiD正信号创建正栅极，但缺少DiD信号的样品也必须用于验证正栅不包含任何DiD阴性样本。
需要滴定以优化荧光信号
在进行完整实验之前，必须优化各种条件，包括细胞染色期间使用的荧光偶联抗体的浓度和脂质体制备期间使用的脂质染料的浓度。流式细胞仪具有荧光强度的检测上限，因此脂质体中掺入的染料过多会导致通过细胞仪的样品中DiD信号水平无法量化。此外，脂质体中过多的DiD可能导致高水平的非特异性染料转移，这可能会扭曲细胞摄取结果。图3报告了一项实验的结果，在该实验中，滴定脂质染料的浓度以确定在使用流式细胞仪检测范围内产生最佳信号的浓度。这是在最终实验感兴趣的组织上进行的:血液（图3A），腹股沟脂肪SVF（图3B）和附睾脂肪SVF（图3C）。选择用于测试的浓度为每1 mL脂质体10mg DiD（高，红色），1mg DiD（中，蓝色）或0.1mg DiD（低，灰色）。脂质体中使用的最高浓度过高，在所有三种组织中都超过了细胞仪的可定量范围（图3A\u2012C，红色）。最低浓度的DiD显示出一些信号（图3 A\u2012C，灰色），但没有观察到PBS处理的细胞之外的清晰群体（图3A\u2012C，黑色）。量化时，每种组织和浓度的DiD MFI的算术平均值表明PBS对照与DiD的中等浓度之间存在明显区别（图3D）。因此，如协议所示，我们选择了中间浓度（图3，蓝色）用于我们的脂质体制备。
使用多抗体组合可以鉴定不同细胞亚群对脂质体的摄取
使用表3中概述的面板，用针对巨噬细胞，B细胞，T细胞，树突状细胞，单核细胞和内皮细胞标记物的抗体对细胞进行染色（图4）。每种组织类型需要略有不同的门控策略，但可以在每种组织中鉴定出大多数相同的细胞类型。一些例外包括内皮细胞，这通常不在血液中发现，以及单核细胞，它们在血液中的频率通常高于其他组织。一旦确定了群体，就可以量化每个细胞群的总大小及其DiD +的频率。可以进行进一步的计算来表征DiD +群体:DiD +细胞中巨噬细胞，内皮细胞等的百分比是多少。请注意，这些是示例门控策略，但不是分析样本的唯一方法。分析将由所选的试剂盒和可用的流式细胞仪决定。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61585/61585fig01.jpg"> 图1:制备的脂质体的示例特征。 （A）如上所述测量大小和zeta电位，并以表格形式报告。每个参数都表示为平均值±标准差。（B）冷冻电镜用于对制备的脂质体进行成像。白色比例尺的长度为 50 nm。（C）在该制备中，使用DLS生成脂质体直径的直方图。该图改编自Osinski等人3。<a href="https://www.jove.com/files/ftp_upload/61585/61585fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61585/61585fig02.jpg"> 图2:来自PBS或脂质体处理小鼠的代表性DiD染色。 （A）PBS和脂质体处理的实验示意图。PBS或脂质体在一周内注射三次。在治疗的第8天收获组织。（乙、丙）代表性流图显示脂质体处理的（C）小鼠的DiD染色阳性，但未显示PBS处理的（B）小鼠的DiD染色阳性。FSC，前向散射。 <a href="https://www.jove.com/files/ftp_upload/61585/61585fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61585/61585fig03.jpg"> 图 3:脂质体中 DiD 的滴定。 用三种不同浓度的DiD制备脂质体并注射到小鼠体内。灰色表示每 1 mL 脂质体 0.1 mg DiD 的低浓度，蓝色表示 1 mg DiD/mL 脂质体的中等浓度，红色表示 10 mg DiD/mL 脂质体的高浓度。使用PBS处理的小鼠作为阴性对照（黑色）。注射后24小时收获血液（A，圆形），腹股沟脂肪（B，三角形）和附睾脂肪（C，方形），并进行处理以分离单细胞悬液。这些样品在流式细胞仪上运行至可检测的DiD水平。呈现每个治疗组叠加的组织特异性直方图，以显示每个浓度的荧光强度（A\u2012C）。还量化了每种组织和浓度的DiD的算术平均值，并绘制了（D）。SSC = 侧向散射。 <a href="https://www.jove.com/files/ftp_upload/61585/61585fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61585/61585fig04v4.jpg"> 图 4:脂肪 SVF、血液和脾脏中细胞亚群的代表性流式细胞术分析。 （A\u2012C）用于识别脂肪SVF（A），脾脏（B）和血液（C）中的细胞亚群和DiD +细胞的门控策略的示意图。缩写:FSC = 前向散射;LD = 活/死;L-DC = 淋巴样树突状细胞;M-DC = 髓系树突状细胞;SSC = 侧向散射。该图改编自Osinski等人3。<a href="https://www.jove.com/files/ftp_upload/61585/61585fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>控制</td>
			<td>目的</td>
		</tr><tr><td rowspan="5">用PBS或生理盐水处理的小鼠</td>
			<td>使用该小鼠的细胞进行以式细胞术对照:</td>
		</tr><tr><td>1. 未染色细胞</td>
		</tr><tr><td>2. 活/死单污渍</td>
		</tr><tr><td>3. 用全面板染色的细胞，但缺乏脂质体荧光，无法在分析过程中确定阳性脂质体信号</td>
		</tr><tr><td>这/这些小鼠/小鼠也将用于确定脂质体在体内是否有任何影响，因为您将在实验中具有非脂质体对照。</td>
		</tr><tr><td>卸载的脂质体</td>
			<td>如果您在脂质体中加载化合物，则应在没有该化合物的情况下合成部分脂质体批次。这解释了脂质体单独对体内的任何影响。</td>
		</tr><tr><td>单独使用迪德</td>
			<td>由于DiD也可以被细胞膜吸收，因此分配一些小鼠以等于脂质体中发现的量接受游离染料将有助于解释任何背景膜染色。</td>
		</tr><tr><td>荧光负一 （FMO） 控制</td>
			<td>这些细胞被面板中除一种抗体以外的所有抗体染色。与上面框中的#3一样，这有助于在分析过程中确定该抗体的真阳性信号</td>
		</tr></tbody></table>表 1:要在此协议中使用的控件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>溶液</td>
			<td>组件</td>
			<td>每批/小鼠所需的近似体积</td>
		</tr><tr><td colspan="3">脂质体制备</td>
		</tr><tr><td>醋酸钙</td>
			<td>1 M 乙酸钙在 H2O</td>
			<td>50毫升</td>
		</tr><tr><td>HEPES缓冲液</td>
			<td>10 mM HEPES H2O，pH 7.4</td>
			<td>50毫升</td>
		</tr><tr><td>赫佩斯的特萨格利塔扎</td>
			<td>在 10 mM HEPES 中</td>
			<td>10毫升</td>
		</tr><tr><td colspan="3">组织采集、处理和染色</td>
		</tr><tr><td>磷酸盐缓冲溶液</td>
			<td>137 mM 氯化钠、2.7 mM 氯化钾、10 mM Na 2 HPO 4、1.8 mM KH 2 PO4 蒸馏 H 2O溶液</td>
			<td>2毫升</td>
		</tr><tr><td>PBS-肝素</td>
			<td>0.1 mM 肝素在 PBS 中</td>
			<td>10毫升</td>
		</tr><tr><td>HEPES缓冲液</td>
			<td>20 毫米丙二醇中的 HEPES</td>
			<td>5毫升</td>
		</tr><tr><td>消化缓冲液</td>
			<td>HEPES 缓冲液中的 2 mg/mL I 型胶原酶</td>
			<td>5毫升</td>
		</tr><tr><td>AKC裂解缓冲液</td>
			<td>0.158 米 NH3厘升， 10 毫米氢氧化钾3， 0.1 毫米钠 2 EDTA 在 ddH 2 O， pH7.2中</td>
			<td>15毫升</td>
		</tr><tr><td>FACS 缓冲液</td>
			<td>1% BSA， 0.05% NaN3 在 PBS 中</td>
			<td>15毫升</td>
		</tr><tr><td>Fc 块（稀释）</td>
			<td>FACS 缓冲液中的 1:50 Fc 块</td>
			<td>250 微升</td>
		</tr><tr><td>固定缓冲液</td>
			<td>PBS中2%多聚甲醛</td>
			<td>200 微升</td>
		</tr></tbody></table>表 2:要准备的解决方案。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>一个</td>
			<td>B</td>
			<td>C</td>
			<td>D</td>
		</tr><tr><td colspan="4">细胞外染色（2x 抗体混合物）</td>
		</tr><tr><td>抗原</td>
			<td>荧光基团</td>
			<td>每 100 μL 测试的 Ab 体积</td>
			<td>所需总量:</td>
		</tr><tr><td>CD45</td>
			<td>全氯乙烯</td>
			<td>0.5 微升</td>
			<td>C 列 x 1.2 x 总计 # 个样本</td>
		</tr><tr><td>CD11b</td>
			<td>全氯乙烯5.5</td>
			<td>0.25 微升</td>
			<td>（0.5 μL/测试） x （1.2） x （# 样品）</td>
		</tr><tr><td>F4/80</td>
			<td>PE Cy7</td>
			<td>0.25 微升</td>
			<td>（0.25 μL/测试） x （1.2） x （# 样品）</td>
		</tr><tr><td>CD19</td>
			<td>聚乙烯-CF594</td>
			<td>1 微升</td>
			<td>（0.25 μL/测试） x （1.2） x （# 样品）</td>
		</tr><tr><td>CD3</td>
			<td>FITC</td>
			<td>1 微升</td>
			<td>（1.0 μL/测试） x （1.2） x （# 样品）</td>
		</tr><tr><td>CD31</td>
			<td>BV605</td>
			<td>0.25 微升</td>
			<td>等。。。</td>
		</tr><tr><td>CD11c</td>
			<td>APC ef780</td>
			<td>1 微升</td>
			<td></td>
		</tr><tr><td>CD115</td>
			<td>体育</td>
			<td>1.5 微升</td>
			<td></td>
		</tr><tr><td colspan="4">要创建抗体混合物，请将 D 列中计算的抗体与 FACS 缓冲液或亮紫染色缓冲液*合并至最终体积（50 μL x 1.2 x 总 # 样品）</td>
		</tr><tr><td colspan="4">活/死染色 （1x）</td>
		</tr><tr><td>生/死</td>
			<td>荧光基团</td>
			<td>每 200 uL 测试的 L/D 体积</td>
			<td>所需总量:</td>
		</tr><tr><td>生/死</td>
			<td>水</td>
			<td>0.67 微升</td>
			<td>C 列 x 1.2 x 总计 # 个样本</td>
		</tr><tr><td colspan="4">细胞内染色 （1x）</td>
		</tr><tr><td>抗原</td>
			<td>荧光基团</td>
			<td>每 50 μL 测试的 Ab 体积</td>
			<td>所需总量:</td>
		</tr><tr><td>αSMA</td>
			<td>FITC</td>
			<td>0.125</td>
			<td>C 列 x 1.2 x 总计 # 个样本</td>
		</tr><tr><td colspan="4">*如果您的组合中使用了多种与亮紫荧光团偶联的抗体，则应使用亮紫染色缓冲液。</td>
		</tr></tbody></table>表 3:用于流式染色的示例抗体组合和染色混合物的计算。

Watch this Scientific Journal Video about Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes at JoVE.com

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

该协议的目标是合成荧光标记的脂质体，并使用流式细胞术在细胞水平上鉴定脂质体的体内定位。

荧光染料标记脂质体体内组织特异性细胞摄取的制备、给药和评估

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...

preparation-administration-assessment-vivo-tissue-specific-cellular

Research

JoVE Journal

Bioengineering

4.0K 次观看.  University of Virginia. 该协议的目标是合成荧光标记的脂质体，并使用流式细胞术在细胞水平上鉴定脂质体的体内定位。

视频: Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...