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 克链霉素 (2 毫克/毫升最终), 和2.5 毫克庆大霉素 (5 µg/毫升最终)。添加 RPMI 1640 至500毫升。使用氢氧化钠调整 ph 值为 7.5 (在调整前, 缓冲器呈黄色/橙色, pH 值约为 6.0)。使用0.45 µm 过滤器进行杀菌消毒。整除和贮存在-20 摄氏度, 可达1年或4摄氏度, 可达1月。</li>
	</ol>
</li>
	<li>
HEPES-碳酸氢钠缓冲器, 10x
	<ol>
<li>
混合23.8 克 HEPES (100 mM 决赛), 21 克 NaHCO 3 (250 毫米决赛), 并将水添加到1000毫升。用 HCl 将 pH 值调整为 7.2. 使用0.45 微米过滤器进行杀菌消毒。在室温下贮存。pH 值随时间变化。定期重新调整 pH 值。</li>
	</ol>
</li>
	<li>
收获媒介 (~ 1 bottle/2 小鼠)
	<ol>
<li>500毫升 RPMI 1640, 加入25毫升热灭活胎牛血清 (5% 最后), 和5毫升 100× HGPG。贮存6月至4摄氏度。</li>
	</ol>
</li>
	<li>
Dithioerythritol (DTE) 解决方案 (40 毫升/小肠)
	<ol>
<li>混合50毫升10×汉克斯平衡盐溶液, Ca2+-和 Mg2+无, 50 毫升 10× HEPES-碳酸氢钠缓冲, 50 毫升热灭活胎牛血清 (10% 最后)。添加350毫升 H2O 和15.4 毫克 dithioerythritol/100 mL (1 毫米决赛)。在使用前准备新鲜的。</li>
	</ol>
</li>
	<li>
乙二胺四乙酸 (EDTA) 溶液 (50 毫升/小肠)
	<ol>
<li>混合50毫升10×汉克斯平衡盐溶液, Ca2+-和 Mg2+-免费与5毫升 100× HGPG。添加445毫升 H2o 添加260µL 0.5 M EDTA/100 mL (1.3 毫米决赛)。在使用前准备新鲜的。</li>
	</ol>
</li>
	<li>
胶原酶溶液 (30 毫升/LP)
	<ol>
<li>到500毫升 RPMI, 添加50毫升血清 (10% 血清最后), 5 毫升 100× HPGP, 1 毫升0.5 米氯化镁2 (1 毫米决赛) 和1毫升 0.5 M CaCl2 (1 毫米决赛)。添加胶原酶, I 型 (100 U/毫升最终)。在使用前准备新鲜的。</li>
	</ol>
</li>
	<li>
密度梯度 (DG) 解决方案
	<ol>
<li>通过混合90毫升的 dg 库存解决方案 (参阅材料表) 与10毫升的 10× PBS 一起准备 1× dg 库存解决方案。保持不育的4摄氏度。</li>
		<li>通过混合44毫升 1× DG 股票溶液和56毫升 RPMI 1640, 准备 44% DG 溶液 (8 毫升/IEL, 8 毫升/LP)。在使用前准备新鲜的。</li>
		<li>通过混合67毫升 1× DG 股票溶液和33毫升 RPMI 1640, 准备 67% DG 溶液 (5 毫升/IEL, 5 毫升/LP)。在使用前准备新鲜的。</li>
	</ol>
</li>
</ol>2. 肠系膜淋巴结、肠道和 Peyer 斑块的分离
<ol><li>弄死小鼠使用二氧化碳麻醉和继发性颈椎脱位。将鼠标放在背上, 用70% 乙醇喷雾。用剪刀做中线切口, 打开皮肤和腹壁, 露出腹腔。</li>
	<li>找到盲肠并使用镊子轻轻拉盲肠下来。使用镊子小心地将小肠移动到右侧, 露出整个 MLN 链, 它与结肠对齐。 注: 一张肠系膜淋巴结链和肠道淋巴引流图以前被描述为13。</li>
	<li>识别链中最靠近盲肠的底部节点。使用一组钳来抓住它周围的肠系膜/脂肪, 并轻轻地删除 MLN 链从底部到顶端通过钳法脂肪从周围的淋巴结与另一套钳。</li>
	<li>将 MLN 链放在用收获介质浸湿的纸巾上。通过滚动纸塔上的链条, 用两套镊子将脂肪拔开, 以去除肠系膜/脂肪。将 MLN 放在冷收获介质中, 以便以后隔离步骤。 注: (i) 不鼓励直接处理节点。相反, 抓住他们周围的肠系膜/脂肪。(二) 尽可能多地切除肠系膜/脂肪, 以改善细胞的生存能力是至关重要的。</li>
	<li>用剪刀切开幽门括约肌和盲肠以上的小肠。使用一组钳将小肠从回肠缓慢拉出, 并使用另一组钳去除附加的肠系膜/脂肪。</li>
	<li>将肚腑放在纸巾上, 用两套镊子蘸上一条沾有收割介质的纸巾, 并戏弄其余的肠系膜/脂肪。 注: (i) 一定要尽可能多地去除肠系膜/脂肪, 以达到最佳的细胞产量和生存能力。(ii) 定期应用收获培养基, 使肠道湿润, 并采取以下步骤。</li>
	<li>收集 Peyer 的补丁, 从肠道取出他们的弯曲剪刀。如果需要, 使用解剖范围或放大镜 (初学者);大多数 Peyer 的补丁应该是可见的训练有素的眼睛。从小肠收集 5-11 (典型) Peyer 的补丁。将 Peyer 的补丁放在冷收割介质中, 以便以后隔离步骤。 注意: Peyer 的斑块是小的, 主要是圆形突起在外肠壁对面的肠系膜附件。</li>
	<li>使用钳的平边和轻轻地从十二指肠向回肠滑动, 以排出粪便内容和粘液。重复一次以去除大部分粘液。 注: 不完全清除粘液可降低细胞活力和产量。不过, 一定要轻轻滑动小肠, 以避免剥离绒毛或破坏基底膜。</li>
	<li>在一个点上使用钝端的细剪刀, 将钝端插入肠道, 并从十二指肠向回肠纵向切开肠道。侧面切开被打开的肚腑入 ~ 2 cm 片断。将肠道部分放置在50毫升圆锥管中, 25 毫升冷收获介质, 用于以后的隔离步骤。 注意: 把剪刀沾上介质会帮助他们毫不费力地滑过肠道。</li>
</ol>3. 从感应部位分离淋巴细胞
<ol><li>使用3毫升注射器的柱塞, 通过70µm 细胞过滤器轻轻游离, 将淋巴细胞从 MLN 中分离出来。用5毫升的冷收获介质冲洗细胞过滤器。</li>
	<li>颗粒 MLN 细胞由离心在 400 x g 5 分钟在4°c。并用重悬1毫升冷收获介质中的细胞颗粒。使用台盼蓝排除计数可行细胞。 注: 红细胞溶解缓冲液可用于去除红细胞中的出血。</li>
	<li>通过在5毫升 prewarmed 胶原酶溶液的15毫升锥形管中孵化 Peyer 的斑块, 在37摄氏度和220转30分钟内, 从 Peyer 的斑块中分离出淋巴细胞。</li>
	<li>漩涡为十五年代在最大设置和过滤器被消化的组织和上清入50毫升圆锥管通过一个70µm 细胞滤网。用3毫升注射器的柱塞轻轻地将剩余的组织块分开, 用5毫升的冷收获介质冲洗细胞过滤器。</li>
	<li>颗粒 Peyer 的补丁细胞由离心在 400 x g 5 分钟在4°c 和并用重悬细胞颗粒在1毫升冷收获媒介。使用台盼蓝排除计数可行细胞。 注意: Peyer 的斑块分离可能有一些污染的固有的细胞和上皮内淋巴细胞。</li>
</ol>4. 小肠上皮内淋巴细胞的分离
<ol><li>用25毫升的冷收获培养基将小肠洗净三次, 通过将管反转10次, 让肠道部分沉淀, 并从上清中倾泻而出。 注意: 组织件应容易沉淀到管子的底部。如果他们不这样做, 这表明太多的肠系膜/脂肪仍然附着或与气泡有关。</li>
	<li>将20毫升的 prewarmed DTE 溶液添加到含有肠道块的50毫升圆锥管中, 并转移到含有搅拌棒的50毫升渗锥形瓶中。搅拌在37°c 和 220 rpm 上的磁力搅拌器20分钟. 使用大磁铁在瓶的外部举行搅拌杆和转移组织和解决方案回到50毫升圆锥管。</li>
	<li>涡流管在最大设置为十年代. 通过70µm 细胞过滤器将上清液转移到新的50毫升锥形管中, 小心地将组织片放在原管中。不要丢弃上清;上清液含有上皮内淋巴细胞。颗粒细胞通过离心在 400 x g 5 分钟在4°c。在10毫升的冷收获介质中并用重悬细胞颗粒, 储存在冰上。</li>
	<li>将20毫升的 prewarmed DTE 溶液添加到含有肠道片的50毫升圆锥管中, 然后转回到含有搅拌棒的50毫升渗锥形瓶中。搅拌在37°c 和 220 rpm 上的磁力搅拌器20分钟. 使用大磁铁在瓶的外部举行搅拌杆和转移组织和解决方案回到50毫升圆锥管。 注意: DTE 是一种能丰富上皮内淋巴细胞的还原剂。</li>
	<li>涡流管在最大设置为十年代. 将上清液通过70µm 细胞过滤器转移到前一管, 其中包含第一个 DTE 治疗的上清液 (从步骤 4.3)。 注意: 其余的肠块用于分离固有层的淋巴细胞。质量控制: 一个组织可以切除和检查组织学, 以确保上皮细胞存在, 基底膜是完整的。</li>
	<li>颗粒细胞通过离心上清液在 400 x g 5 分钟在4摄氏度。并用重悬细胞颗粒在8毫升 44% DG 溶液室温 (RT)。</li>
	<li>将8毫升 44% DG 溶液/电池悬浮液转移到17毫米直径 x 100 mm 型14毫升聚丙烯圆底管。衬底与5毫升 RT 67% DG 溶液。离心机在 1600 x 克20分钟的 RT 不使用刹车。 注: 可以用牛血清预处理导管, 防止细胞黏附在管壁上。</li>
	<li>请注意, 在44% 到67% 界面上, 存活的细胞形成一条带 (粘液), 死细胞和一些上皮细胞位于梯度顶端的一层膜中, 红血球在颗粒中。通过真空吸尘, 将渐变的顶层移至界面的2厘米以内。使用巴斯德吸管在界面上收获菲比涂层, 并将其转移到含有40毫升冷收获介质的50毫升圆锥管上。</li>
	<li>颗粒细胞通过离心在 400 x g 5 分钟在4°c。并用重悬1毫升冷收获介质中的细胞颗粒。使用台盼蓝排除计数可行细胞。</li>
</ol>5. 小肠内固有细胞的分离
<ol><li>将25毫升的 prewarmed EDTA 溶液加入含有剩余肠道的烧瓶中, 去除肠道组织块中的上皮细胞。搅拌瓶在37°c 和 220 rpm 上的磁力搅拌器30分钟. 使用大磁铁在瓶的外部举行搅拌杆, 并小心地丢弃上清, 同时保持肠块内的瓶子。 注: (i.) EDTA 是一种钙离子螯合剂, 它靶向钙依赖的结点, 并促进肠道上皮细胞的脱离。(二) 上清应该是多云的, 并可用于隔离 IEC, 如果他们需要收集。</li>
	<li>重复步骤5.1。 注: 此孵化后上清液应少多云。质量控制: 一个组织可以切除和检查组织学, 以确保肠道上皮细胞已被删除, 基底膜是完整的。从上清液中抽取的样品也可以通过流式细胞仪进行评估, 以确认白细胞 (CD45+细胞) 不存在。</li>
	<li>在烧瓶中加入50毫升的 RT 收获培养基。用磁铁简单搅拌。让肠道块安定下来, 小心地丢弃上清。 注: 这一步骤确保在胶原酶消化前, 通过螯合钙对胶原酶的作用至关重要的钝化酶去除 edta。</li>
	<li>添加30毫升的 prewarmed 胶原酶溶液的烧瓶, 并在37°c 和 220 rpm 在一个磁性搅拌器45分钟搅动肠道部分。</li>
	<li>通过70µm 细胞过滤器, 将消化组织和上清液转移到50毫升锥形管中。使用3毫升注射器的柱塞, 通过过滤器, 轻轻地分离剩余的组织块。用10毫升的冷收获介质冲洗过滤器。</li>
	<li>颗粒细胞通过离心在 400 x g 5 分钟在4°c。并用重悬8毫升环境温度 44% DG 溶液中的细胞颗粒。</li>
	<li>将8毫升 44% DG 溶液/电池悬浮液转移到17毫米直径 x 100 mm 型14毫升聚丙烯圆底管。衬底与5毫升的环境温度 67% DG 溶液。离心机的梯度在室温和 1600 x g 20 分钟, 没有刹车。</li>
	<li>用真空吸尘去除上面的脂肪层。使用巴斯德吸管在界面上收获菲比涂层, 并将其转移到含有40毫升冷收获介质的50毫升圆锥管上。</li>
	<li>颗粒细胞通过离心在 400 x g 5 分钟在4°c。并用重悬1毫升冷收获介质中的细胞颗粒。使用台盼蓝排除计数可行细胞。</li>
</ol>

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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. 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=Antigen-driven+induction+of+CD11c+on+intestinal+intraepithelial+lymphocytes+and+CD8++T+cells+in+vivo.">Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8+ T cells in vivo.</a> J Immunol. 154 (11), 5684-5693 (1995).</li><li>Van Damme, N., 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=Chemical+agents+and+enzymes+used+for+the+extraction+of+gut+lymphocytes+influence+flow+cytometric+detection+of+T+cell+surface+markers.">Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers.</a> J Immunol Methods. 236 (1-2), 27-35 (2000).</li></ol>

作者没有什么可透露的。

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。

<table><tbody><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Sigma-aldrich</td><td>G3126-100G</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Gentamicin</td><td>Life Technologies</td><td>15710-072</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>S318-500</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>21870-076</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Fisher Scientific</td><td>S233-500</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>26140-079</td><td></td></tr><tr><td>10x Hanks&#039; balanced salt solution</td><td>Sigma-aldrich</td><td>H4641-500ML</td><td></td></tr><tr><td>1,4-Dithioerythritol</td><td>Sigma-aldrich</td><td>D9680-5G</td><td></td></tr><tr><td>0.5M EDTA, pH 8.0</td><td>Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>Calsium chloride hexahydrate</td><td>Sigma-aldrich</td><td>21108-500G</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-aldrich</td><td>M2670-100G</td><td></td></tr><tr><td>Collagenase, Type I</td><td>Life Technologies</td><td>17100-017</td><td></td></tr><tr><td>DG gradient stock solution (Percoll)&amp;nbsp;</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>70-&amp;micro;m cell strainer&amp;nbsp;</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>14 mL Polypropylene Round-Bottom Tube</td><td>Corning</td><td>352059</td><td></td></tr><tr><td>Erlenmeyer flask&amp;nbsp;</td><td>Kimble</td><td>26500R-50mL</td><td></td></tr><tr><td>Magnetic stirrer</td><td>Thermo Fisher</td><td>50094596</td><td></td></tr><tr><td>Stir bar</td><td>Fisher Scientific</td><td>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 ...

isolating-lymphocytes-from-the-mouse-small-intestinal-immune-system

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

52.4K 次观看.  Stony Brook University. 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。

视频: Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

科研

生物学

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

从小鼠的补丁中提取体液免疫的关键参与者: 先进和优化的淋巴细胞分离协议

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

癌症研究

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

免疫与感染

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

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

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

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 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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 microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

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 ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

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

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 antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

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 ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.