Name: isolation of lamina propria mononuclear cells from murine colon using collagenase e
Uploaded: 2019-09-26T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E at JoVE.com

这项工作得到了#1K08HL088260和#1R01HL133462-01A1（NHLBI）（A.B.P.，H.N.，S.J.）和巴切洛尔儿科研究基金会（D.M.，H.N.，S.J.，A.A.H.，A.B.P.）的支持。本研究中使用的C57BL/6和BALB/c小鼠要么在我们的工厂中繁殖，要么由杰克逊实验室或塔科尼奇提供。

所有研究均根据迈阿密大学米勒医学院机构动物护理和使用委员会（IACUC）审查和批准的啮齿动物研究规程进行，该委员会符合美国协会制定的兽医标准实验室动物科学（AALAS）。
1. 准备解决方案
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如表1所述，制备结肠缓冲液、硅基密度分离介质100%、硅基密度分离介质66%、硅基密度分离介质44%、胶原酶E消化缓冲液和FACS缓冲液。
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<li>在手术前一天准备?...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+the+intestinal+tract:+pathogenesis+and+treatment.">Inflammation in the intestinal tract: pathogenesis and treatment.</a> Digestive diseases. 27 (4), 455-464 (2009).</li><li>Pabst, R., Russell, M. 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(132), e57281 (2018).</li><li>Weigmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subsequent+analysis+of+murine+lamina+propria+mononuclear+cells+from+colonic+tissue.">Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue.</a> Nature Protocols. 2, 2307-2311 (2007).</li><li>Bull, D. M., Bookman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+characterization+of+human+intestinal+mucosal+lymphoid+cells.">Isolation and functional characterization of human intestinal mucosal lymphoid cells.</a> Journal of Clinical Investigation. 59 (5), 966-974 (1977).</li><li>Davies, M. D., Parrott, D. 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此视觉协议描述了用于分离结肠单核细胞（包括拉米那体淋巴细胞 （LPL）的耐受性好的方法。鉴于此方案在评估严重的移植后小鼠结肠炎模型时进行了优化，其中炎症细胞因子和组织损伤导致恢复的MNC生存能力较差，我们预计这些方法可以转化为其他需要结肠MNC的型态分析的应用程序。这些包括，但不限于评估炎症性肠病小鼠模型中的结肠炎症，研究结肠炎靶向治疗的免疫反应，以及传染性病原...

虽然胃肠道 （GI） 主要致力于处理和重新吸收来自食物的营养物质，但胃肠道也保持在血管、淋巴和神经系统以及许多其他器官的完整性中的核心作用。其粘膜和亚粘膜免疫系统1。GI免疫系统在胃肠道和全身健康中都具有影响作用，因为它经常接触来自食物、共体细菌或入侵病原体1、2的外来抗原。因此，GI免疫系统必须保持一个微妙的平衡，在其中它容忍非致病性抗原，同时适当响应致病性抗原1，2。当耐受性和防御的平衡被打乱时，局部或全身免疫失调和炎症可能导致无数的疾病1，2，3。
肠中至少有70%的淋巴细胞在体内4。大多数初级免疫学相互作用涉及肠道中三个免疫站中的至少一个：1）佩耶尔的贴片，2）皮内淋巴细胞（IEL）和3）拉米纳表体淋巴细胞（LPL）。其中每一个都由一个复杂的免疫细胞互联网络组成，对肠道5的正常免疫挑战作出快速反应。限于肌肉粘膜上方的频闪，松散的层状膜是肠道粘膜的结缔组织，包括软体、血管、淋巴排水和粘膜神经系统的脚手架，以及许多与生俱来和自适应免疫子集6，7，8，9。LPL由CD4+和CD8+T细胞组成，近似比例为2：1，血浆细胞和骨髓体细胞包括，树突状细胞、乳腺细胞、嗜酸性细胞和巨噬细胞6。
人们越来越有兴趣了解肠道的免疫调节和炎症，因为它涉及到各种疾病状态。克罗恩病和溃疡性结肠炎等疾病都表现出不同程度的结肠炎10，11，12。此外，接受异体骨髓移植（Allo-BMT）的骨髓或免疫系统的恶性或非恶性疾病患者可以发展各种形式的结肠炎，包括1）从调理方案直接毒性在BMT之前，2）由BMT和3）移植-宿主疾病（GVHD）后由供体型T细胞驱动，在BMT13、14、15之后对组织中的供体异抗原作出反应，由免疫抑制引起的感染。所有这些后BMT并发症导致肠道的免疫环境显著改变16，17，18。该方法允许对小鼠结肠中的免疫细胞积累进行可靠的评估，在BMT之后应用于小鼠受体时，有助于对参与移植耐受性中的供体和受体免疫细胞进行有效检测19 ，20.肠道炎症的其他原因包括恶性肿瘤、食物过敏或肠道微生物群的中断。该协议允许从结肠进入肠道单核细胞，并经修改后，在临床前小鼠模型中访问小肠的白细胞。
使用搜索词"肠道和免疫细胞和分离"的PubMed搜索揭示了200多个出版物，描述了小肠消化提取免疫细胞的方法。然而，对结肠的类似文献搜索没有产生指定免疫细胞与结肠分离的精心划定的协议。这可能是因为结肠有更肌肉和间质层，使其更难完全消化比小肠。与现有协议不同，该协议专门使用不含其他细菌胶原酶（胶原酶 D/胶原酶 I）的胶原酶 E。我们证明，使用该协议，可以在保持分离的肠道单核免疫细胞（MNC）质量的同时，实现结肠组织的消化，而无需添加抗结块试剂，如醋酸钠（EDTA）、Dispase II和脱氧核糖核酸I（脱氧核糖核酸I）21，22，23 。该协议经过优化，允许从鼠结肠中可重复的强健提取可存活的MNC，以便进一步定向研究，并应有助于研究结肠的免疫学或（有修改）小肠24，25.

<table>
	<tbody>
		<tr>
			<td>60 mm Petri DIsh</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Lonza BioWhittaker</td>
			<td>BW17-517Q</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Disposable Serological Pipette</td>
			<td>Corning</td>
			<td>4100</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Non-Sterile Conical Tubes</td>
			<td>TruLine</td>
			<td>TR2002</td>
			<td></td>
		</tr>
		<tr>
			<td>18- gauge Blunt Needle</td>
			<td>Becton Dickinson</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Disposable Serological Pipette</td>
			<td>Corning</td>
			<td>4250</td>
			<td></td>
		</tr>
		<tr>
			<td>40 micrometer pore size Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon Tube</td>
			<td>Corning</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A4503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation Buffer</td>
			<td>Biolegend</td>
			<td>420801</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli Collagenase E from Clostridium histolyticum</td>
			<td>Sigma</td>
			<td>C2139</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M Sterile Solution</td>
			<td>Amresco</td>
			<td>E177-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo /Fisher Scientific -HyCLone</td>
			<td>SV30014.03</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>GE Healthcare-HyClone</td>
			<td>SH30237.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare-Life Sciences</td>
			<td>1708901</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium</td>
			<td>Corning</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>VWR Life Science Amresco</td>
			<td>97064-646</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Lonza BioWhittaker</td>
			<td>17-942E</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of lamina propria mononuclear cells from murine colon using collagenase e

肠道是体内免疫细胞数量最多的家庭。小型和大型肠道免疫系统可调节外源抗原，并调节对强效微生物衍生的免疫刺激的反应。因此，肠道是许多疾病中免疫调节和炎症的主要靶点，包括但不限于炎症性肠病，如克罗恩病和溃疡性结肠炎、骨后移植物与宿主疾病 （GVHD）骨髓移植（BMT），和许多过敏和传染性疾病。胃肠道炎症和结肠炎的Murine模型被大量用于研究胃肠道并发症和临床前优化预防和治疗策略。通过对肠道免疫细胞的分离和型板分析从这些模型中收集的数据对于进一步了解可应用于改善胃肠道和全身炎症疾病的免疫理解至关重要。本报告描述了使用混合硅基密度梯度界面将单核细胞（MNC）从结肠分离的高效方案。该方法可重复分离大量可行的白细胞，同时最大限度地减少污染碎片，从而允许随后通过流式细胞仪或其他方法进行免疫型型。

当使用鼠结肠疾病模型时，能够量化和定性评估结肠的MNC中涉及炎症过程的多个免疫细胞子集是有帮助的。通过应用该协议获得的MNC的单细胞悬浮液，有助于以稳健且可重复的方式进行表型表征。作为在各种实验环境下应用这种分离方法的原理证明，我们使用这种方法检索了结肠MNC，并在从小鼠身上分离的细胞上进行了多参数流细胞测定（图1?...

Watch this Scientific Journal Video about Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E at JoVE.com

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

该协议的目标是通过使用胶原酶对组织进行酶消化，分离位于结肠层状的单核细胞。该协议允许对单核细胞进行有效分离，从而产生单个细胞悬浮液，进而可用于强健的免疫分体。

使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

使用本协议对肠道免疫细胞进行表型分析的分离已证明适用于对GI和全身性炎症性疾病的理解。这种方法通过最大限度地减少污染碎片，通过Fleur的阿托米斯特里或其他方法进行后续免疫表型，分离出大量可行的单核细胞。由于肠道是许多疾病炎症的主要靶点，此协议可能有助于研究小鼠癌症、过敏、移植和自身免疫模型中的肠道免疫群。为了优化本协议的产量和效率，建议提前准备关键解决方案和材料。对鼠标进行安乐死并进行垂直中线切口后，使用精细的解剖剪刀打开内膜。使用钳子，将小肠移动到一侧，并公开下降结肠。在下降的结肠上稍微向上拉，以最大地暴露结肠的直肠部分。然后，切开骨盆深处的远毛直肠，将整个结肠解剖为从远毛直肠到切毛帽的一个单元。首先，将结肠放在湿润的纸巾上，用一把剪刀或钳子的钝端对肠壁施加轻度压力，并提取固体凳子。接下来，将结肠放在培养皿中，使用10毫升注射器，用18个量表的钝填充针头用10毫升的冷冻结肠缓冲液冲洗肠道。将结肠放在装满 5-10 毫升冷冻结肠缓冲液的培养皿中，并手动搅拌以清洗剩余的结肠内。重复此洗涤 2-3 次，使用新的培养皿，每次洗涤都含有新鲜缓冲液。在此之后，将结肠转移到含有新鲜冷冻结肠缓冲液的新培养皿中。将结肠纵向从其肌肉更发达的直肠端切到近端结肠，以生成单个矩形开放结肠片。丢弃盘中的中位数，用干净的冷冻结肠缓冲液替换。将矩形结肠组织放在纸巾上，用结肠缓冲液润湿，然后水平切开，然后切成小块。使用细钳将结肠碎片收集到含有 20 毫升冷冻结肠缓冲液的 50 毫升聚丙烯锥形管中。然后，通过大力旋转管子 30 秒来清洗碎片。洗涤后，让组织碎片沉淀到管底，然后或吸尘，确保避免失去组织碎片，每次洗涤使用新鲜结肠缓冲液重复整个洗涤过程 3 次。首先，在含有洗涤结肠片段的管子中加入20毫升的胶原酶消化缓冲液。密封管，并在37摄氏度的轨道摇床中放置，旋转速率为2倍G，旋转60分钟。确保组织片段在搅拌过程中保持恒定运动。首先，将 5 毫升 66%硅基密度分离介质倒入 3 个单独的 15 毫升聚丙烯管中。接下来，使用25毫升血清移液器只收集从胶原蛋白消化的上流水液1。通过 40 微升滤液纤维滤芯过滤器过滤上清液，放入清洁的 50 毫升聚丙烯锥形管中。将管子完全填充冰镇结肠缓冲液，以淬火胶原酶消化缓冲液。然后旋转细胞，吸上一液，并保持在冰上的颗粒。继续执行文本协议中概述的胶原蛋白酶消化 2。胶原蛋白消化2完成后，通过18规格的钝端针在管子和10毫升注射器之间大力冲洗组织片段。重复此冲洗至少 7-8 通道，一直持续到看不到毛组织碎片或脱布里。接下来，通过一个40微米差的过滤织物细胞过滤器，将组织分解悬浮液放入一个干净的50毫升聚丙烯管中。将管子填充到边缘，用冷却结肠缓冲液在4摄氏度下以800次G加热和离心5分钟。通过真空吸气丢弃上经剂，将胶原蛋白消化液消化 1 中重新悬浮的颗粒拉到相应的管中。在此之后，将每个颗粒重新悬浮在 24 毫升 44%硅基密度分离介质中。每个结肠。使用 10 毫升血清移液器将该介质的 8 毫升缓慢分层到包含 66% 硅基密度分离介质的 3 根管上。使用称重刻或平衡器小心平衡离心桶内的管。在20摄氏度的859倍G下离心20分钟，不休息。在取出管子之前，让棒子完成休息，注意不要破坏梯度界面的细胞。首先，可视化 5 毫升标记附近的梯度接口，通常存在 1-2 毫升厚的白色带。真空吸气并丢弃梯度前 7 毫升，以便更方便地接触移液器。使用连续的手动吸力和稳定旋转的手腕运动，收集细胞的界面层。收集，直到两个梯度之间的接口是清晰和折射，并转移接口到一个新的50毫升聚丙烯圆锥形管。接下来，将 FACS 缓冲液添加到此浴缸，直到总悬浮液达到 50 毫升。在 4 摄氏度下在 800 倍 G 下离心 5 分钟。然后，通过真空吸气吸上经液，并在1毫升的FACS缓冲液中重新悬浮颗粒。本视频中描述的程序可用于从结肠中分离单核细胞，或者通过修改从小肠分离出单核细胞。此外，在使用胶原蛋白酶E或D进行分离时，使用非DNA酶1治疗，进行细胞切除术和数据分析，以比较凋亡和坏死淋巴细胞的分数。继附件5和可修复活死蓝色染料染色单细胞悬浮液后，每次分离后，与没有DNAse的胶原酶D相比，分离后没有DNAse的胶原酶E显示的活细胞比例要高得多。即使与DNA酶的胶原酶相比。此外，我们在胶原酶E组中以41.0%的中位数百分比识别坏死细胞，而在胶原蛋白D组中，这一比例为90.0%。75.9%在胶原酶E DNAse组和80.3%在胶原酶DDNAse组。然后，在收到 BMT 或异构或合成 BMT 模型后，在第 7 天将多近位流量细胞学应用于从 CD45.2 气门-C 接收小鼠分离的 MNC。计算和比较了从BMT接受者结肠中提取的捐赠者CD4阳性和CD8阳性T细胞的均数。因此，在这样的小鼠模型中识别和/或量化稀有免疫细胞种群非常重要。稀有子集，包括供体衍生的FOX-P3阳性T调控细胞在合成和异体BMT模型中进行评估。使用此方法，甚至可以分析罕见的子集，如供体派生结肠T调控渗透接受者小鼠结肠后，BMT。应该注意的是，胶原酶E在37摄氏度下是活跃的。因此，所有胶原酶溶液应预先加热，以充分胶原酶活性，并彻底淬火以终止活性。该协议允许大量的单核细胞，可用于后续表征的弗勒尔细胞学，分子生物学技术，显微镜，培养，和位点，等等。该协议提供了一个可靠的评估炎症在实验鼠与受控小鼠在骨髓移植模型。从而促进移植耐受性新型调节免疫机制的发现。来自梭菌的胶原蛋白酶是一种应采用实验室安全技术处理的试剂。吸入后会对健康造成危害，并可能导致皮肤和眼睛刺激。

Research

JoVE Journal

Immunology and Infection

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

大脑和脊髓的单核细胞的分离，使用Percoll梯度

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Isolation, Processing and Analysis of Murine Gingival Cells

小鼠牙龈细胞的分离，处理和分析

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

从大鼠睾丸支持细胞的分离和管周细胞

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

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 ...