Name: flow cytometric analysis for identification of the innate and adaptive immune cells of murine lung
Uploaded: 2021-11-16T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung at JoVE.com

这项工作得到了NIH拨款R01CA238263和R01CA229784（VAB）的支持。

该协议中描述的所有研究和实验都是根据Beth Israel Deaconess医疗中心的机构动物护理和使用委员会（IACUC）的指南进行的。使用六到十周龄的C57BL / 6小鼠，无论男女，都用于开发该协议。
1. 手术切除和组织准备
<ol><li>通过腹腔内注射1mL三溴乙醇（根据标准方案制备; 材料表）。 注意：在肺部研究中应避免CO2 窒息，因为它可能导致肺损伤并改变肺免疫细?...

<ol>
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肺免疫细胞的鉴定可能具有挑战性，因为与驻留在其他组织中的对应物相比，肺中存在多种免疫细胞类型及其独特的免疫表型特征。在几种病理条件下，具有不同表型特征的细胞出现在肺部。例如，博来霉素诱导的肺损伤导致肺泡腔中循环单核细胞来源的巨噬细胞募集，在那里它们可以停留长达一年，甚至在博来霉素诱导的纤维化后持续存在。与组织驻留的AM相反，循环的单核细胞衍生巨噬细胞?...

鼠呼吸道含有独特的免疫系统，负责对抗病原体和维持免疫稳态。肺免疫系统由在表型、功能、起源和位置方面具有显著异质性的细胞群体组成。常驻肺泡巨噬细胞 （AMs） 主要起源于胎儿单核细胞，位于肺泡腔1，而骨髓来源的间质巨噬细胞 （IM） 位于肺实质2。IM可以通过CD206的表达式进一步细分。CD206+ IM位于支气管周围和血管周围区域，而CD206-IM位于肺泡间质3。最近提出了IM的几个子分类3，4，5，6。虽然IM的研究少于AM，但最近的证据表明它们在调节肺免疫系统中起着至关重要的作用7。此外，CD206还以替代激活的AM8表示。
肺树突状细胞（DC）是肺免疫细胞的另一组异质性，就其功能特性，位置和起源而言。在肺中已经描述了四种DC亚类：常规CD103 + DC（也称为cDC1），常规CD11b + DC（也称为cDC2），单核细胞衍生的DC（MoDC）和浆细胞样DCs9，10，11，12，13。前三个子类可以定义为主要的组织相容性复合物（MHC）II + CD11c + 9，10，14，15。浆细胞样 DC 表达 MHC II，CD11c 中度阳性，但表达高水平的 B220 和 PDCA-19，13，16。在幼稚的鼠肺中，CD103 DC和CD11b DC位于气道间质中，而浆细胞样DC位于肺泡间质17中。
单核细胞的两个主要群体在稳态下驻留在肺中：经典单核细胞和非经典单核细胞。经典的单核细胞是Ly6C+ ，对最初的炎症反应至关重要。相比之下，非经典单核细胞是Ly6C- ，并被广泛视为抗炎细胞3，16，18。最近，描述了另一个CD64 + CD16.2 + 单核细胞群体，其起源于Ly6C- 单核细胞并产生CD206 + IMs3。
嗜酸性粒细胞主要出现在蠕虫感染或过敏性疾病期间的肺部。然而，在稳定状态期间，肺实质中存在少量嗜酸性粒细胞，称为常驻嗜酸性粒细胞。与常驻嗜酸性粒细胞相反，炎症性嗜酸性粒细胞存在于肺间质和支气管肺泡灌洗 （BAL） 中。在家尘螨（HDM）小鼠模型中，炎症性嗜酸性粒细胞在抗原介导的刺激后被招募到肺部。有人提出，居民嗜酸性粒细胞可能通过抑制对HDM19的T辅助剂2（Th2）致敏而在过敏中具有调节作用。
与其他肺髓细胞相反，中性粒细胞表达Ly6G但不表达CD68，其特征在于CD68-Ly6G +免疫表亲型的特征16，20，21。可视化研究表明，在稳态期间，肺在血管内隔室中保留了一池嗜中性粒细胞，并容纳了相当数量的血管外嗜中性粒细胞22。与嗜酸性粒细胞类似，在稳定状态下的BAL中找不到嗜中性粒细胞;然而，几种形式的免疫刺激，如LPS激发，哮喘或肺炎，驱动嗜中性粒细胞进入肺泡腔，导致它们存在于BAL21，22，23中。
大量肺 CD45+ 细胞代表自然杀伤 （NK）、T 细胞和 B 细胞，大多数骨髓标志物均为阴性24。在幼稚小鼠的肺部，可以根据CD11b和MHC II18的表达来鉴定这三种细胞类型。大约25%的肺CD45 +细胞是B细胞，而NK细胞在肺部的百分比高于其他淋巴和非淋巴组织24，25，26。在肺T细胞中，相当一部分是CD4-CD8-，在呼吸道感染中起重要作用26。
由于肺宿主非常复杂和独特的免疫系统，因此已经开发出几种用于识别肺免疫细胞的门控策略并报告了16，18，20，27。本文描述的门控策略提供了一种全面且可重复的方法，使用9种标志物鉴定多达12种不同的肺髓系和非髓系免疫群体。已使用其他标记来验证结果。此外，还提供了一种详细的方法来制备单细胞悬浮液，该单细胞悬浮液可最大限度地减少细胞死亡，并允许鉴定肺免疫细胞区室的最完整特征。应该注意的是，肺非免疫细胞的鉴定，如上皮细胞（CD45-CD326 + CD31-），内皮细胞（CD45-CD326-CD31 +）和成纤维细胞需要不同的方法28，29。此处描述的协议和方法不包括此类人群的识别。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL syringe plunger</td>
			<td>EXELINT</td>
			<td>26265</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G needles</td>
			<td>BD Precision Glide Needle</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needles</td>
			<td>BD Precision Glide Needle</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Falcon</td>
			<td>3520</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>ThermoFisher</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Falcon/corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>ThermoFisher</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD11b</td>
			<td>Biolegend</td>
			<td>101215</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117339 / 117337</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103115</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD64</td>
			<td>Biolegend</td>
			<td>139319</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse CD68</td>
			<td>Biolegend</td>
			<td>137009</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse GR-1</td>
			<td>Biolegend</td>
			<td>108433</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>anti-mouse Siglec F</td>
			<td>Biolegend</td>
			<td>155503</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>AVERTIN</td>
			<td>Sigma-Aldrich</td>
			<td>240486</td>
			<td></td>
		</tr>
		<tr>
			<td>B220</td>
			<td>Biolegend</td>
			<td>103228</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CD103</td>
			<td>Biolegend</td>
			<td>121405 / 121419</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>CD24</td>
			<td>Biolegend</td>
			<td>138503</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Biolegend</td>
			<td>100205</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>CX3CR1</td>
			<td>Biolegend</td>
			<td>149005</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore Sigma</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123133</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>FcBlock (CD16/32)</td>
			<td>Biolegend</td>
			<td>101301</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>R&#38;D Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Serrated Forceps</td>
			<td>Roboz Surgical Instrument Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td>ThermoFisher</td>
			<td>00-5523-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Futura Safety Scalpel</td>
			<td>Merit Medical Systems</td>
			<td>SMS210</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Fixable Far Read Dead Cell Stain Kit</td>
			<td>ThermoFisher</td>
			<td>L34973</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>MERTK</td>
			<td>Biolegend</td>
			<td>151505</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>MHC-II</td>
			<td>Biolegend</td>
			<td>107621</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>NK1.1</td>
			<td>Biolegend</td>
			<td>108705</td>
			<td>For details see Table 2</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>VWR</td>
			<td>Model 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated benchtop centrifuge</td>
			<td>SORVAL ST 16R</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small curved scissor</td>
			<td>Roboz Surgical Instrument Co</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometric analysis for identification of the innate and adaptive immune cells of murine lung

呼吸道与外部环境直接接触，需要精确调节的免疫系统来提供保护，同时抑制对环境抗原的不良反应。肺宿主着几个先天性和适应性免疫细胞群，这些免疫细胞提供免疫监视，但也介导保护性免疫反应。这些细胞保持健康的肺免疫系统平衡，也参与几种病理状况，如哮喘，感染，自身免疫性疾病和癌症。表面和细胞内蛋白质的选择性表达为肺的免疫细胞提供独特的免疫表型特性。因此，流式细胞术在稳态和病理条件下鉴定此类细胞群方面起着重要作用。本文提出了一种方案，描述了一种一致且可重复的方法，用于在稳态条件下鉴定驻留在健康小鼠肺部的免疫细胞。然而，该协议也可用于识别各种疾病模型中这些细胞群的变化，以帮助识别肺免疫景观中的疾病特异性变化。

门控策略 我们的门控策略的第一步是排除碎屑和双层（图1A）。仔细排除双联对于避免假阳性人群至关重要（补充图S2）。然后，使用CD45 +鉴定免疫细胞，CD45 +是造血细胞的标志物（图1B）。可以添加活死染色剂以排除死细胞。然而，该协议导致<5%的CD45 + 细胞死亡（图1C），而更多的CD45...

Watch this Scientific Journal Video about Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung at JoVE.com

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

在这项研究中，我们提出了一种有效且可重复的方案来分离小鼠呼吸系统的免疫群体。我们还提供了一种方法，使用基于9色的流式细胞术面板来鉴定驻留在健康小鼠肺部的所有先天性和适应性免疫细胞。

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

由于肺宿主非常复杂和独特的免疫系统，因此已经开发并报告了几种用于识别肺免疫细胞的门控策略。由于存在几种不同的方法，因此很难比较不同实验室产生的结果。我们的门控策略提供了一种全面且可重复的方法，使用九种标志物鉴定多达12种不同的肺髓样和非髓系免疫群体。本方案描述了稳态条件下肺免疫群体的表征和鉴定。然而，该协议可用于识别各种疾病模型中这些细胞群的变化，其中它可以帮助识别肺免疫景观的疾病特异性变化。首次使用这种技术的研究人员应该注意消化条件和试剂，因为它们对免疫细胞从肺组织中的释放有很大的影响。首先准备安乐死动物进行手术。通过在四肢上使用针头或胶带来稳定小鼠背侧，然后使用70%乙醇对腹侧区域的皮肤进行消毒。从颈部到腹部做一个切口。从胸部区域去除皮肤，以及肋骨和胸骨。使用18至21号针将10毫升冷PBS注射到右心室中，直到它们完全变白，从而冲洗肺部。然后，切除胸腺和心脏而不接触肺部。将肺与周围组织分离，并将其转移到含有冷BSA缓冲液的管中。将肺转移到培养皿中，用两把细手术刀切碎，然后将其放入50毫升的锥形管中。加入五毫升消化缓冲液以洗涤平板。盖上管盖，在37摄氏度下以每分钟150次旋转的速度在眼眶摇床上消化肺部30分钟。通过加入10毫升冷BSA缓冲液停止反应。消化后，使用18号针头混合并溶解肺片。将70微米过滤器放在新的50毫升锥形管的顶部，并将消化的肺混合物转移到过滤器中。使用10毫升注射器柱塞的橡胶侧粉碎过滤器上剩余的肺片，并用BSA缓冲液清洗。在7摄氏度下以350 G离心单细胞悬浮液八分钟。弃去上清液并将细胞重悬于一毫升ACK裂解缓冲液中。使用一毫升移液管混合悬浮液，并在室温下孵育90秒。向反应混合物中加入10毫升冷的BSA缓冲液，并在4摄氏度下以350 G离心7分钟。弃去上清液，将沉淀重悬于染色缓冲液中，并使用血细胞计数器计数细胞。以每毫升500万个细胞的浓度重悬细胞以进行表面染色。将每孔200微升中的100万个细胞转移到96孔板中，并在4摄氏度下以350 G离心板7分钟。通过在染色缓冲液中稀释抗1632抗体来制备流式细胞术阻滞溶液。将细胞重悬于50微升流式细胞术阻断溶液中。将悬浮液在4摄氏度或冰上孵育15至20分钟。然后，向板中加入150微升染色缓冲液，并在4摄氏度下将其350G离心五分钟。通过稀释染色缓冲液中的表面抗体来制备表面抗体溶液。将细胞重悬于50微升的表面抗体溶液中，并在黑暗中以4摄氏度孵育板。然后，用染色缓冲液洗涤细胞两次。通过混合三组份固定和透化稀释剂和一份染色缓冲液来制备固定和透化缓冲液。将细胞重悬于96孔板每孔孔的50微升制备缓冲液中，并在黑暗中以4摄氏度孵育20至25分钟。用纯化的去离子水稀释透化缓冲液，制备一次透化缓冲液，并用它来洗涤细胞。通过用一毫升通透缓冲液稀释来制备细胞内抗体溶液。将细胞重悬于96孔板每孔50微升稀释的细胞内抗体溶液中，并在黑暗中以4摄氏度孵育40分钟。用透化缓冲液洗涤细胞，然后用染色缓冲液洗涤细胞。最终洗涤后，将细胞重悬于200微升染色缓冲液中。在流式细胞仪上采集每个样品至少150万个细胞。在手术成功和适当的术后手术后，通过门控策略排除了碎片和双层。通过流式细胞术使用CD45 +造血标记物鉴定免疫细胞。这些细胞被区分为活的或死的。免疫肺细胞使用抗GR-1和抗CD68抗体分为三组。使用Ly6G作为唯一标记物鉴定和验证中性粒细胞。CD45 +群还含有自然杀伤细胞，T细胞和B细胞。这些细胞通过自然杀伤1.1，CD3和B220抗体染色和验证。支气管肺泡灌洗物鉴定出CD11b标志物阳性的嗜酸性粒细胞，以及不表达高水平CD11b的肺泡巨噬细胞。这些细胞对CD103和CD11b标志物表现出阳性或阴性反应。CD103阴性细胞根据CD64表达分为常规和单核细胞来源的树突状细胞。单核细胞来源的树突状细胞CD24树突状标志物阳性率低，CD64泛巨噬细胞标志物阳性。基于CD64和GR-1表达对具有经典和非经典单核细胞的间质巨噬细胞进行区分。这些细胞对CX3C趋化因子受体一的表达呈阳性。该过程中最重要的步骤是适当的组织收获，消化和单细胞悬浮液的制备，以及对活细胞和单线态的门控。除了通过流式细胞术表征肺的免疫细胞外，还可以在分离的细胞群中进行细胞培养和功能测定。这项技术可以为研究人员探索肺部疾病的新问题铺平道路，例如感染，自身免疫性疾病和癌症。

Research

JoVE Journal

Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and Identification of Extravascular Immune Cells of the Heart

心脏外免疫细胞的分离与鉴定

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...