Name: isolating lymphocytes from the mouse small intestinal immune system
Uploaded: 2018-02-28T15:00:00
Description: Watch this Scientific Journal Video about Isolating Lymphocytes from the Mouse Small Intestinal Immune System at JoVE.com

学理学士由 NIH 赠款 (R01 AI076457) 和石溪大学提供的资金支持。Z.Q. 得到 NIH 赠款 (K12 GM102778) 的支持。

所有动物实验都是按照国家卫生研究院的指导方针进行的, 并经石溪大学机构动物护理和使用委员会批准。
注: 确保在执行程序之前授予所有批准。
1. 解决方案准备
<ol><li>
HGPG (HEPES, l-谷氨酰胺, 青霉素/链霉素, 庆大霉素), 100×
	<ol>
<li>混合59.6 克 HEPES (500 毫米最后), 14.6 克 l-谷氨酰胺 (200 毫米最终), 1×106 U 青霉素 (2000 U/毫升最终), 1 克?...

<ol>
	<li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat Rev Immunol. 10 (3), 159-169 (2010).</li><li>Brandtzaeg, P., Kiyono, H., Pabst, R., Russell, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminology:+nomenclature+of+mucosa-associated+lymphoid+tissue.">Terminology: nomenclature of mucosa-associated lymphoid tissue.</a> Mucosal Immunol. 1 (1), 31-37 (2008).</li><li>Lefrancois, L., Lycke, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+small+intestinal+intraepithelial+lymphocytes,+Peyer's+patch,+and+lamina+propria+cells.">Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells.</a> Curr Protoc Immunol. , 19 (2001).</li><li>Sheridan, B. 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(111), (2016).</li><li>Koscso, B., Bogunovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+Purification+of+Mouse+Intestinal+Dendritic+Cell+and+Macrophage+Subsets+by+Flow+Cytometry.">Analysis and Purification of Mouse Intestinal Dendritic Cell and Macrophage Subsets by Flow Cytometry.</a> Curr Protoc Immunol. 114, 11-14 (2016).</li><li>Goodman, T., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+gamma-delta+T-cell+receptor+on+intestinal+CD8++intraepithelial+lymphocytes.">Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes.</a> Nature. 333 (6176), 855-858 (1988).</li><li>Huleatt, J. W., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta2+integrins+and+ICAM-1+are+involved+in+establishment+of+the+intestinal+mucosal+T+cell+compartment.">Beta2 integrins and ICAM-1 are involved in establishment of the intestinal mucosal T cell compartment.</a> Immunity. 5 (3), 263-273 (1996).</li><li>Sheridan, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammadelta+T+cells+exhibit+multifunctional+and+protective+memory+in+intestinal+tissues.">gammadelta T cells exhibit multifunctional and protective memory in intestinal tissues.</a> Immunity. 39 (1), 184-195 (2013).</li><li>Sheridan, B. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymph+nodes+draining+the+small+intestine+and+colon+are+anatomically+separate+and+immunologically+distinct.">The lymph nodes draining the small intestine and colon are anatomically separate and immunologically distinct.</a> Mucosal Immunol. 9 (2), 468-478 (2016).</li><li>Lorenz, R. G., Newberry, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+lymphoid+follicles+can+function+as+sites+for+induction+of+mucosal+immune+responses.">Isolated lymphoid follicles can function as sites for induction of mucosal immune responses.</a> Ann N Y Acad Sci. 1029, 44-57 (2004).</li><li>Kim, S. K., Reed, D. S., Heath, W. R., Carbone, F., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+migration+of+CD8+T+cells+in+the+intestinal+mucosa.">Activation and migration of CD8 T cells in the intestinal mucosa.</a> J Immunol. 159 (9), 4295-4306 (1997).</li><li>Huleatt, J. 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本文提出了一种从肠道黏膜诱导 (MLN 和 PP) 和效应器 (LP 和 IEL 室) 中分离淋巴细胞的详细协议。该议定书已制定, 以平衡投入 (时间) 和产出 (可行性和产量), 以最大限度地提高生产率和成果。该协议还确保了 LP 和 IEL 隔间之间的最小交叉区划污染。
从小鼠小肠中分离免疫细胞的几项协议已发布3,4,5,<...

胃肠道有许多褶皱和突起, 代表着分离内部身体和外部环境的最大界面。肠道免疫系统在维持胃肠道屏障功能方面起着至关重要的作用。它经常接触到膳食抗原、共生细菌和致病微生物。因此, 它必须保持对食物抗原和共生细菌的耐受力, 同时保持对致病微生物的有效免疫反应的能力 (1)。肠道免疫系统可以解剖划分为诱导点, 在那里天真的淋巴细胞由抗原呈现细胞激活, 携带抗原从肠道粘膜, 和效应部位, 其中活化淋巴细胞发挥特定效应函数2。诱导部位包括 Peyer 斑块 (PP) 的组织淋巴结构, 通过专门的 M 细胞和区域引流肠系膜淋巴结 (MLN) 的作用直接调查肠道腔。效应部位由固有层 (LP) 组成, 它是基底膜下面的结缔组织, 肠上皮, 位于基底膜上方的一个细胞, 含有上皮内淋巴细胞 (IEL)。淋巴细胞是适应性免疫的主要参与者, 它可以调节对感染和癌症的保护, 也可能导致炎症性疾病的免疫病理。研究这些明显的解剖黏膜隔室中的淋巴细胞, 以更好地了解它们的诱导和效应功能, 具有重要的意义。
由于探索肠道中发生的免疫事件的调查人员数量正在加速, 因此需要一个相对简单和统一的隔离这些隔间淋巴细胞的协议。几个研究小组发布了一些协议, 它们共享几个类似的过程, 用于隔离来自小鼠小肠隔间的免疫细胞3,4,5,6,7.但是, 它们之间存在着一些技术上的差异, 这取决于个别协议的重点。例如, 重点是将免疫细胞从 LP 中分离出来, 一项协议检查了各种酶消解对细胞活力、细胞表面标记表达和孤立免疫细胞组成的影响5。另一项协议突出了一种快速、可再生的方法, 用于分离无密度离心的淋巴细胞6。最后, 还存在特定的协议, 目的是从小肠的不同组织层分离单个核吞噬7。在这里, 一个高度可重复性的协议, 允许从 MLN, PP, LP, 和 IEL 室的小肠的顺序隔离淋巴细胞的数量。
我们专注于从 LP 和 IEL 隔间隔离高度纯净的人群, 这些舱基本上没有来自其他肠道的污染物。这种广泛使用的协议在可接受的时间限制范围内产生的最大纯和可行的淋巴细胞的高收益率4,8,9,10,11,12。该协议还确保从 LP 和 IEL 室隔离淋巴细胞与最小的交叉区划污染, 使一个真正的机会来研究这些不同的舱室淋巴细胞。分离的淋巴细胞可以受到进一步的操作, 如流细胞分析或功能分析。该协议已成功地应用于分离的小鼠小肠和结肠在细菌感染, 如李斯特菌, 伤寒沙门氏菌和耶尔森氏杆菌假结核菌淋巴细胞。感染和炎症条件, 如化学和病原体引起的结肠炎。该协议也可用于分离先天免疫细胞, 如树突状细胞, 巨噬菌, 中性粒蛋白, 单核, 从小鼠小肠和结肠。

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">HEPES</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:119px;">BP310-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine&#160;</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">G3126-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Penicillin-Streptomycin</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Gentamicin</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15710-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S318-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">RPMI 1640</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">21870-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Sodium bicarbonate</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S233-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fetal bovine serum</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">10x Hanks&#39; balanced salt solution</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">H4641-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,4-Dithioerythritol</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">D9680-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.5M EDTA, pH 8.0</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calsium chloride hexahydrate</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td>21108-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td>M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase, Type I</td>
			<td style="width:123px;">Life Technologies</td>
			<td>17100-017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DG gradient stock solution (Percoll)&#160;</td>
			<td style="width:123px;">GE Healthcare</td>
			<td style="width:119px;">17-0891-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Red Blood Cell Lysis Buffer</td>
			<td style="width:123px;">Biolegend</td>
			<td style="width:119px;">420301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#181;m cell strainer&#160;</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 mL Polypropylene Round-Bottom Tube</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erlenmeyer flask&#160;</td>
			<td style="width:123px;">Kimble</td>
			<td style="width:119px;">26500R-50mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Magnetic stirrer</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:119px;">50094596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Stir bar</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">14-512-148</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating lymphocytes from the mouse small intestinal immune system

肠道免疫系统在维持胃肠道屏障功能方面起着至关重要的作用, 通过对膳食抗原和共生细菌进行耐受性反应, 同时对致病进行有效的免疫应答。微生物.此外, 很明显, 局部肠道免疫对远程和全身免疫具有深远的影响。因此, 研究如何诱发肠道免疫应答以及反应的免疫学结果是很重要的。在这里, 描述了一个详细的协议, 用于隔离小肠诱导部位的淋巴细胞, 如肠道相关淋巴组织 Peyer 的补丁和引流肠系膜淋巴结和效应部位, 如固有层和肠上皮。这项技术确保从小肠组织中分离出大量的淋巴细胞, 具有最佳的纯度和生存能力, 并且在可接受的时间限制内区划污染最小。从肠道组织中分离淋巴细胞和其他免疫细胞的技术能力使人们能够理解对胃肠道感染、癌症和炎症性疾病的免疫反应。

描述了协议的示意图表示形式 (图 1)。肠道粘膜诱导和效应部位的淋巴细胞有明显的组织。Peyer 的斑块 (PP) 和肠系膜淋巴结 (MLN) 在组织良好的 T 细胞区和 B 细胞卵泡中含有淋巴细胞, 而肠道上皮则含有更弥漫分布的淋巴细胞。固有层 (LP) 包含弥漫分布淋巴细胞和淋巴细胞, 如分离的淋巴滤泡 (ILF) 和 cryptopatches 的组织淋巴结构中。每个肠道免疫室也具?...

Watch this Scientific Journal Video about Isolating Lymphocytes from the Mouse Small Intestinal Immune System at JoVE.com

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

小鼠小肠免疫系统淋巴细胞的分离

在这里, 我们描述了一个详细的协议, 从诱导部位分离淋巴细胞, 包括肠道相关淋巴组织 Peyer 的补丁和引流肠系膜淋巴结, 和效应部位, 包括固有层和小肠上皮的小肠免疫系统。

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Research

JoVE Journal

Immunology and Infection

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

科研

免疫与感染

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

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 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 ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

小鼠肾上腺的分离、固定和免疫荧光成像

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...