Name: high-dimensionality flow cytometry for immune function analysis of dissected implant tissues
Uploaded: 2021-09-15T15:00:00
Description: Watch this Scientific Journal Video about High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues at JoVE.com

这项研究得到了美国国立卫生研究院校内研究计划的部分支持，包括国家生物医学成像和生物工程研究所。 免责声明：美国国立卫生研究院、其官员和员工不推荐或认可任何公司、产品或服务。

注： 图1 概述了流式细胞术方案。
1）试剂制备
<ol><li>准备用于稀释酶和组织培养的培养基。<ol><li>将 5 mL 4-（2-羟乙基）-1-哌嗪乙磺酸 （HEPES） 缓冲溶液加入 500 mL RPMI 培养基中并摇匀。将培养基储存在4°C直至进一步使用。</li>
	</ol></li>
	<li>计算酶溶液的体积。 注：酶溶液的体积是含有酶（胶原酶和DNase I）的培养基的体积，该酶在6孔板中消化切块组...

<ol>
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本综述描述了一种从生物材料植入物中分离细胞以获得均匀细胞悬液的详细方法。此外，还提供了用于多色流式细胞术的细胞悬液染色的详细方案，以及配置流式细胞仪以获得最佳结果的步骤。细胞分离方法可能涉及多个步骤，通常利用手动组织解剖，然后用蛋白水解酶进行酶消化，以解离组织中的细胞外基质并破坏细胞-细胞连接，从而将单个细胞从组织中释放出来。消化后，需要进一步处理，...

医学领域的进步导致频繁使用植入材料来支持受损组织的功能或再生长 1,2。这些设备包括起搏器、重建美容植入物和用于骨折固定的骨科板等设备 3,4。然而，用于制造这些植入物的材料及其植入位置在决定这些植入物的成功方面起着重要作用5,6,7。作为异物，这些植入物可以产生来自宿主的免疫反应，从而导致排斥或耐受8.这一因素推动了生物材料研究，以产生能够在植入后吸引所需免疫反应的材料 9,10,11,12。
免疫反应是再生医学领域的基本要求，在再生医学领域，组织或器官在实验室的生物材料骨架（支架）周围生长，以替换受损的组织或器官13,14,15,16。在再生医学中，目标是通过使用细胞、信号和支架来替换缺失或受损的组织，其中每个细胞都可以通过免疫反应极大地调节17。此外，即使需要缺乏免疫反应，也很少缺乏免疫活性，而不是存在所需的调节谱18。流式细胞术等技术可以在表征对用于涂覆植入装置或开发组织工程支架的各种生物材料的免疫反应模式方面发挥重要作用19。
反过来，这些信息最终将有助于开发免疫系统可以很好地耐受的植入物的生物材料，或者开发可以在组织工程中发挥建设性作用的支架。正确制备用于流式细胞术分析的样品是避免通过荧光激活细胞分选进行免疫表征结果不准确的重要步骤20,21。因此，本综述提出了一种详细的方法，可用于从支架组织中分离细胞、对细胞悬液进行染色和流式细胞术分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Plate</td>
			<td>Fisher Scientific</td>
			<td>07-000-646</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Brilliant Stain Buffer Plus</td>
			<td>BD Biosciences</td>
			<td>566385</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix</td>
			<td>BD Biosciences</td>
			<td>554655</td>
			<td>For only fixing cells</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A7906</td>
			<td>For preparing FACS staining buffer</td>
		</tr>
		<tr>
			<td>CD11b AF700</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>Clone: M1/70</td>
		</tr>
		<tr>
			<td>CD11c PerCP/Cy5.5</td>
			<td>Biolegend</td>
			<td>117325</td>
			<td>Clone: N418</td>
		</tr>
		<tr>
			<td>CD197 PE/Dazzle594</td>
			<td>Biolegend</td>
			<td>120121</td>
			<td>Clone: 4B12</td>
		</tr>
		<tr>
			<td>CD200R3 APC</td>
			<td>Biolegend</td>
			<td>142207</td>
			<td>Clone: Ba13</td>
		</tr>
		<tr>
			<td>CD206 PE</td>
			<td>Biolegend</td>
			<td>141705</td>
			<td>Clone: C068C2</td>
		</tr>
		<tr>
			<td>CD45 BUV737</td>
			<td>BD Biosciences</td>
			<td>612778</td>
			<td>Clone: 104/A20</td>
		</tr>
		<tr>
			<td>CD86 BUV395</td>
			<td>BD Biosciences</td>
			<td>564199</td>
			<td>Clone: GL1</td>
		</tr>
		<tr>
			<td>CD8a BV421</td>
			<td>Biolegend</td>
			<td>100737</td>
			<td>Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Comp Bead anti-mouse</td>
			<td>BD Biosciences</td>
			<td>552843</td>
			<td>For compensation control</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore Sigma</td>
			<td>11284932001</td>
			<td>Bovine pancreatic deoxyribonuclease I (DNase I)</td>
		</tr>
		<tr>
			<td>F4/80 PE/Cy7</td>
			<td>Biolegend</td>
			<td>123113</td>
			<td>Clone: BM8</td>
		</tr>
		<tr>
			<td>Fc Block</td>
			<td>Biolegend</td>
			<td>101301</td>
			<td>Clone: 93</td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Solution Kit</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>For fixing and permeabilization of cells.</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td>Buffer to supplement cell media</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Millipore Sigma</td>
			<td>5401127001</td>
			<td>Blend of purified Collagenase I and Collagenase II</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
			<td>Thermo Fisher</td>
			<td>L23105</td>
			<td>Viability dye</td>
		</tr>
		<tr>
			<td>Ly6c AF488</td>
			<td>Biolegend</td>
			<td>128015</td>
			<td>Clone: HK1.4</td>
		</tr>
		<tr>
			<td>Ly6g BV510</td>
			<td>Biolegend</td>
			<td>127633</td>
			<td>Clone: 1A8</td>
		</tr>
		<tr>
			<td>MHCII BV786</td>
			<td>BD Biosciences</td>
			<td>742894</td>
			<td>Clone: M5/114.15.2</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Thermo Fisher</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermo Fisher</td>
			<td>11875176</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Siglec F BV605</td>
			<td>BD Biosciences</td>
			<td>740388</td>
			<td>Clone: E50-2440</td>
		</tr>
		<tr>
			<td>V-bottom 96-well plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-dimensionality flow cytometry for immune function analysis of dissected implant tissues

在个体体内植入实验室培养的组织或医疗设备的成功取决于受体宿主的免疫反应。将植入物视为异物，敌对和失调的免疫反应可能导致植入物的排斥反应，而调节反应和稳态的恢复可能导致其接受。分析在 体内 或 离体 环境中解剖的植入物的微环境有助于了解免疫反应的模式，这最终有助于开发新一代生物材料。流式细胞术是一种众所周知的技术，用于根据细胞表面标志物表征免疫细胞及其亚群。本综述描述了一种基于手动切割、酶消化和通过细胞过滤器过滤的方案，用于从解剖的植入物组织中分离均匀的细胞悬浮液。此外，还解释了多色流式细胞术染色方案，以及初始流式细胞仪设置的步骤，以通过流式细胞术表征和定量这些分离的细胞。

用于免疫分析的流式细胞术panel的开发过程通常依赖于将结果与现有数据和现场文献进行比较。了解群体在流式细胞术中的呈现方式对于正确解释数据至关重要。无论如何，群体和细胞类型在不同的组织中可能以不同的方式出现，因此可以预料到会有一些变异性。在明确定义的对照组织的背景下，可以针对具有充分研究的细胞类型的已知组织评估这种染色优化。 <strong class...

Watch this Scientific Journal Video about High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues at JoVE.com

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

从解剖的植入物中分离细胞并通过流式细胞术对其进行表征，可以显著有助于了解针对植入物的免疫反应模式。本文描述了一种从解剖植入物中分离细胞及其染色以进行流式细胞术分析的精确方法。

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

该协议可以帮助我们表征宿主对不同生物材料的免疫反应，从而有助于设计更好的未来医疗植入物。流式细胞术为我们提供了有关免疫细胞浸润生物材料的信息，这有助于我们确定细胞如何响应损伤和材料植入的机制，以及改进治疗的靶点。可以修改相同的方案以表征不同环境中的免疫反应。解剖一周前接受ECM植入物的小鼠的股四头肌，并收集在含有5毫升无血清培养基的50毫升管中。最后用剪刀将组织切成小块。接下来，向试管中加入 5 毫升消化酶培养基。将装有消化培养基和切块组织的试管置于 37 摄氏度和 100 RPM 的振荡培养箱中 45 分钟。在孵育结束时，将消化的组织悬浮液通过 70 微米过滤器过滤到单独的 50 毫升管中。使用五毫升注射器头捣碎任何固体组织块，并用室温PBS清洗过滤器。丢弃过滤器中任何剩余的残留物，并用室温 PBS 将管的体积调节至 50 毫升。通过离心收集细胞，并将沉淀重悬于PBS中的10毫升冷的5毫摩尔EDTA溶液中。如果样品中存在血细胞，则将沉淀重悬于一毫升红细胞裂解缓冲液中，孵育 10 分钟，然后加入 9 毫升 EDTA。将悬浮液放在冰上 10 分钟。然后用冷PBS将音量调节至50毫升。通过离心收集细胞并重悬沉淀和 100 微升冷 PBS。将 10 微升悬浮液转移到单独的微量离心管中，并与 10 微升的 trip 和 blue 混合进行计数。将剩余体积的细胞分配到V形底96孔板的每个孔中。从孔中取出 20 微升细胞，并将它们添加到新孔中以用作未染色的对照。然后使用PBS使每个孔中的最终体积达到200微升。通过离心收集板孔底部的细胞，并将细胞重悬于每孔1至1000浓度的100微升活性染料中。在孵育结束时，用 100 微升新鲜 PBS 洗涤每个孔，并将沉淀重悬于每孔 200 微升染色缓冲液中。第二次离心后，将细胞重悬于50μL的1至100稀释的单核细胞阻滞剂中。将细胞悬液在冰上孵育 5 分钟，然后加入 50 微升抗体混合物。在避光的冰上孵育 30 分钟。然后用每孔 100 微升染色缓冲液洗涤孔。再次离心细胞并用 200 微升染色缓冲液洗涤。再重复该过程两次，然后将细胞重悬于 400 微升染色缓冲液中。在分析样品之前，运行未染色的样品，以便在侧向散射图与前向散射图上调整细胞群。接下来，运行染色的样品。提取自发荧光特征。然后导入 FCS 文件以将其用作解压缩控件。单击解调图标以打开解调向导，并通过对正负群体进行选通，使用所有单色控件执行解调。接下来，单击“QC”部分并查看复杂性指数。复杂度指数是衡量光谱特征集合在光谱特征未混合在一起时的可区分程度的指标。最后，通过单击实时解混来解调样品。在这里，可以观察到对照小鼠组织上的14种颜色事实的结果。在该分析中，可以观察到几个游说者群体，例如 ly6g 阳性中性粒细胞和 ly6c 中高表达单核细胞类别。CD11c 高 MHC 两个阳性树突状细胞在针对 CD11b 进行门控以排除巨噬细胞和其他髓系细胞（如中性粒细胞和单核细胞）时很容易显现。通过关注该 CD11c 阳性细胞群，可以鉴定 CD206 阳性 CD86 阳性树突状细胞的子集，其中包括 CD86 高 M1 样树突状细胞和 CD206 高 M2 样树突状细胞群。F4/80 表现出在各种巨噬细胞群体中常见的表达梯度。Siglec-F 存在于 F4-80 阳性和阴性群体中，最有可能分别对应于巨噬细胞亚群和嗜酸性粒细胞。尽管用细胞表面标记物染色可以对细胞类型进行这些指定，但重要的是要注意，表达不同标记物的细胞应以功能性方式观察，而不是更二元分类。尝试此方案时，请记住，在设计检测组合之前了解未染色样品的自发荧光特征是实现更好混合的关键。除了分析细胞外，还可以将分离的细胞分类到特定的群体中，以便通过细胞测定、体外转录组学分析进行下游评估，或进行显微镜分析以评估细胞形态。生物材料流式细胞术分析的复杂性不断增加，有助于弥合基础免疫学研究与新疗法工程之间的差距。

Research

JoVE Journal

Bioengineering

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

科研

生物工程

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

一个手机上的宽视场荧光显微镜和荧光成像流式细胞仪

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

使用光声流细胞测定卵巢癌检测

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...