作者希望承认来自 R37 DK045729 (JLM)、R01 AR060837 (to) 和密歇根大学胃肠研究中心分子核心 P30 DK034933 的支持。

所有动物实验都被密歇根大学的动物使用和照料委员会批准。
1. 无菌聚 d-赖氨酸 (PDL) 和层粘连蛋白溶液的制备
<ol><li>至少在细胞分离前一天, 制备聚 d-赖氨酸 (PDL) 和层粘连蛋白涂层板。 注: 6 井和12井板都是根据实验目标准备的。一般情况下, 用12井板进行定量分析, 采用西方印迹;然而, 6 井板被用来举行蒸压 (无菌) 盖玻片的免疫组化。24-井板用于培养电梯的 Ca2 +通量成像。</li>
	<li>稀释的库存解决方案与超纯组织培养级, 无 DNase 和无 RNase 水在组织培养罩与高效过滤空气。</li>
	<li>
杀菌玻璃盖玻片
	<ol>
<li>用无菌钳盖玻片, 将盖玻片浸泡在100% 乙醇的烧杯中15分钟. 允许乙醇在组织培养罩中蒸发, 然后放入6井板中。</li>
		<li>或者, 将几个盖玻片单独放置在2毫米厚的吸纸上, 然后放在塑料容器中, 然后蒸压釜。</li>
	</ol>
</li>
</ol>2. 涂层板
注意: 在无菌组织培养层流罩下执行这些步骤。
<ol><li>解冻1毫克/毫升 PDL 的股票和稀释1:10 与无菌, 组织培养级水, 使最终浓度为100µg/毫升。用2毫升每井涂层6井板, 1 毫升覆盖一个井12井板和0.5 毫升为一个井在24井板。将剩余稀释的 PDL 存储在12毫升整除数-20 摄氏度。</li>
	<li>在允许 pdl 将水井涂上至少1小时后, 用无菌吸管去除 pdl。使板材在组织培养层流罩下完全风干。 注: 在用无菌吸管去除 PDL 溶液后, 在1周内通过0.22 µm 过滤器并存储在4摄氏度后, 可以再使用两次。</li>
	<li>解冻的层粘连蛋白的股票, 以避免凝胶形成。 注: 未稀释的层粘连蛋白在8摄氏度以上时变为固体。</li>
	<li>稀释层粘连蛋白 (0.5 毫克/毫升) 1:50 与无菌 Dulbecco 的磷酸盐缓冲盐水 (DPBS), 以获得最终浓度10µg/毫升。将稀释的层粘连蛋白储存在12毫升整除数,-20 摄氏度。</li>
	<li>将稀释的层粘连蛋白添加到含干 PDL 的水井中。使用1毫升覆盖6井表面和0.5 毫升覆盖12井板的底部。对于盖玻片, 添加400µL 稀释层粘连蛋白到盖玻片, 以达到表面覆盖。</li>
	<li>如果使用的是同一天 (快速涂层), 则将涂覆的板材在37摄氏度处孵育2小时。如果没有, 密封的塑料包装板, 并存储隔夜在4°c (慢涂层)。 注: 密封是避免干燥和污染的关键。</li>
	<li>用吸管小心地取下层粘连蛋白溶液, 以避免擦伤表面。用无菌 DPBS 轻轻冲洗水井三次。</li>
	<li>将完整的胶质生长介质添加到每个井中, 并保持37摄氏度的板材, 直到准备添加胃癌悬浮液。 注: 聚 d-赖氨酸和层粘连蛋白涂层板可储存3天, 在4摄氏度。将板材提前几天涂上, 然后存放在4摄氏度, 用无菌 DPBS 三次洗盘子。在最后一次洗完后, 将2毫升的无菌 DPBS 添加到每个井中。用 parafilm 封住盘子, 在4摄氏度处贮存。必须确保层粘连蛋白不干燥。</li>
</ol>3. 隔离解决方案的准备工作
<ol><li>准备 EDTA/HEPES/DPBS 离解溶液。对于500毫升的溶液, 使用无菌 DPBS (不含钙和镁), 以最终解决10毫米 HEPES 和5毫米 EDTA。到490毫升的 DPBS, 添加5毫升1米 HEPES 缓冲和5毫升的0.5 米 EDTA 库存解决方案。存储在4°c, 直到使用。</li>
	<li>使用表 1中列出的试剂制备胶质细胞泥沙介质。</li>
	<li>使用表 2中列出的试剂制备胶质生长介质。 注: 含有 GDNF 的胶质生长培养基可贮存4摄氏度的一周。</li>
</ol>4. 切除肠道部分
注意: 在 euthanization 之前, 老鼠被允许自由进入食物和水, 用异氟醚滴方法和切除一个重要器官。C57BL/6 小鼠超过8周的年龄被使用。两种性别在准备过程中没有明显的差异。
<ol><li>用过量的异氟醚弄死老鼠, 将仰卧位放置在解剖托盘中。固定四肢, 用70% 乙醇消毒腹部。用镊子把皮肤顶起来, 然后用剪刀打开腹部。</li>
	<li>抬起肝脏, 辨认远端胃/幽门。然后移除近端肠道的7厘米。小心不要撕掉肠道。</li>
	<li>
用钳夹住幽门, 同时把肠系膜剪掉。
	<ol>
<li>用剪刀后面的钝夹层刮走附着的胰腺。</li>
		<li>将肠道段放入冰冷的 DPBS, 没有 Ca2 +或毫克2 + (图 1A)。 注意: 其他部分的肠道或结肠也可以删除使用相同的技术。</li>
	</ol>
</li>
	<li>使用5或10毫升注射器连接到钝端20克针, 以冲洗出粪便的内容与冰冷 DPBS (图 1B)。</li>
	<li>用史密斯的最初描述的技术将肠道分成3厘米段以去除纵肌. 12和修改在桑德里森等。10。 注意: 每30毫升的 HEPES/EDTA/DPBS 隔离液中, 最多可使用两个小鼠的肠道段。</li>
	<li>断约4厘米, 把木棍从棉花尖上分开。把木棍浸泡在 DPBS (图 1C)。</li>
	<li>将其中一个肠段平滑地滑到润湿棒上 (图 2A-C)。</li>
	<li>用针鼻钳移除附着的肠系膜 (图 2D)。</li>
	<li>
使用干净的剃刀刀片或手术刀, 使两个纵向的缺口沿小肠的肠系膜连接 (图 2E)。
	<ol>
<li>用 DPBS 把棉花尖弄湿。沿肌肉纵向摩擦湿润的棉尖, 松开纵肌/肌间丛丛 (LMMP)。然后在 LMMP 去除过程中, 水平地移动棉花尖, 沿肠段长度从圆形肌肉中 LMMP。</li>
		<li>保持拇指和食指之间的棍子的尖端, 同时稳定的部分与中指和第四手指 (图 2F)。当完成后, LMMP 将很容易剥离肠道部分。</li>
	</ol>
</li>
	<li>除去从肠道管中剥离的 LMMP 并附着在棉签上, 除非需要从肌间丛丛中收获电梯。</li>
	<li>用剃刀刀片从木棍中纵向剥开肠道部分。</li>
	<li>将剥去的肠道组织 (主要是粘膜和黏膜下层加上圆形肌肉) 储存在冰 DPBS 上。重复步骤4.4–4.11 直到所有肠段都准备好。</li>
</ol>5. 上皮黏膜的去除
<ol><li>用细剪刀把肠子切成0.5 厘米的小块。</li>
	<li>将组织放置在50毫升的无菌锥形离心管中, 其中含有30毫升的冰冷 EDTA/HEPES/DPBS 溶液。</li>
	<li>岩石的组织包含溶液在4°c 10 分钟60倾斜每分钟。</li>
	<li>先将 EDTA/HEPES/DPBS 缓冲液吹打一次, 然后用预湿润的吸管将组织和缓冲悬浮液吸出5毫升的塑料吸管, 向上和向下 (研磨) 20 次去除上皮粘膜。</li>
	<li>通过100µm 尼龙细胞过滤器浇注混合物, 从 EDTA 孵化中收集组织。丢弃混浊的粘液填充流 (图 3)。</li>
	<li>将在过滤器中保留的组织放入一个新的50毫升锥形离心管中, 其中含有30毫升的冰冷 EDTA/HEPES/DPBS, 使用针鼻钳。</li>
	<li>重复 EDTA/HEPES/DPBS 孵化第二次通过摇摆组织在4°c 10 分钟60倾斜每分钟, 以剥离额外的上皮粘膜。</li>
	<li>先用缓冲液将5毫升吸管吸干, 然后将组织悬浮液移至20次, 以去除粘液细胞。 注: 随着更多的上皮细胞被移除, 组织将倾向于坚持体内的吸管。</li>
	<li>在第二次孵化后, 通过100µm 尼龙细胞过滤器浇注混合物, 并丢弃流经的组织。第二个流量应该明显少于第一个流 (图 3)。</li>
	<li>重复10分钟 EDTA 孵化, 直到解决方案几乎是明确的 (约3至4孵化取决于组织的数量和研磨的有效性) (图 3)。</li>
</ol>6. 从固有层和黏膜下层收集肠道胶质细胞
<ol><li>将组织从尼龙过滤器转移到15毫升锥形离心管, 其中5毫升的商用细胞回收解决方案和岩石为25–30分钟4摄氏度。</li>
	<li>
Triturate 轻轻地 10x, 将肠道胶质细胞从固有层中分离出来。
	<ol>
<li>过滤通过40µm 过滤器和收集滤液在一个干净的50毫升管。</li>
		<li>用1毫升的 DPBS 将尼龙过滤器上的组织冲洗干净。</li>
		<li>丢弃组织并将5–6毫升的滤液转移到清洁的15毫升锥形离心管中。</li>
		<li>旋转的滤液在 2000 x g在摆动斗离心机5分钟在4摄氏度。</li>
	</ol>
</li>
	<li>在至少1毫升的泥沙缓冲液中, 用500µL 吸管尖轻轻吹打颗粒, 重新悬浮胶质细胞。避免引入气泡。 注: 根据需要调整泥沙体积的井和板数。例如, 1.2 毫升为一个6井板;2.4 毫升为两个6井板;2.4 毫升为一个12井板。</li>
	<li>吸管200µL 细胞悬浮入6井或100µL 的每个井为12个好的 PDL-层粘连蛋白涂层板包含胶质生长媒介。</li>
	<li>在将细胞添加到盘子中并放置在37摄氏度孵化器中后, 请勿打扰菜肴, 以允许最大数量的细胞加入。 注意: 一个健康的细胞准备将附加在 6–8 h, 但等待至少24小时之前, 通过轻轻吹打与非黏附细胞的媒体, 以改变媒体。</li>
	<li>使用单元格进行实验3–4天后, 他们被放置到文化 (图 4)。使用对特定蛋白质标记感兴趣的抗体进行免疫荧光分析 (表 3)。例如, 在这里使用了 GFAP、S100b 和 p75NTR 或 Sox10。e-钙粘蛋白或阿尔法平滑肌肌动蛋白抗体用于评估其他细胞类型的污染程度。</li>
</ol>7. 免疫荧光染色
<ol><li>从文化中吸取媒体, 用 PBS 冲洗两次。</li>
	<li>在室温下固定20分钟的培养基, 4% 多聚甲醛。</li>
	<li>取出固定剂, 然后用 PBS 冲洗3次。</li>
	<li>Permeabilize 细胞与 PBS 含有0.2% 海卫 X-100 在室温下。</li>
	<li>在室温下, 用抗鸡 IgY、抗兔或抗小鼠 IgG 等2小时的阻断溶液孵化透细胞。</li>
	<li>
在一夜之间孵化出主要抗体的细胞,例如, GFAP (1:1, 000)。
	<ol>
<li>用 PBS 冲洗细胞3次。如果执行定位进行步骤7.7。</li>
	</ol>
</li>
	<li>在室温下孵化下一组至少2小时的原发抗体, 但最好在4摄氏度过夜。使用1:1000 稀释兔 anti-S100 或1:500 稀释小鼠 anti-p75NTR。 注意: 所有抗体在 PBS 中被稀释。</li>
	<li>用 PBS 冲洗细胞3次以去除原发抗体。然后用适当的荧光标记的二级抗体在室温下孵育2小时的冲洗细胞 (表 3)。</li>
	<li>用 PBS 冲洗3次以去除二次抗体。</li>
	<li>将盖玻片安装在幻灯片上, 使用带有 DAPI 的载入介质滴, 并在荧光显微镜下查看细胞。</li>
</ol>8. 流式细胞仪
<ol><li>用 DPBS 冲洗细胞一次, 然后添加0.25% 胰蛋白酶-EDTA 每制造商的协议, 删除粘附胶质细胞的流式细胞术。将胰蛋白酶-EDTA 溶液中的细胞孵化为37摄氏度, 3 分钟. 通过在完全胶质细胞生长培养基中收集细胞来终止消化。</li>
	<li>以 400 x g离心为5分钟收集细胞, 然后在 PBS 中重新悬浮细胞。</li>
	<li>修复细胞与4% 多聚甲醛10分钟, 然后 permeabilize 0.2% 海卫 X-100 在 PBS, 如步骤7.4 所述。</li>
	<li>将含有 1% BSA 的 PBS 和组织特异免疫球蛋白的细胞阻断在20分钟内,如驴抗鸡 IgY (IgG) (H + L) (1:1, 000) 或驴抗山羊 IgG (H + L) (1:1, 000)。</li>
	<li>孵育细胞与主要抗体 GFAP (1:2, 000), e-钙黏蛋白 (1:400), α平滑肌动蛋白 (1:500), 或 Pgp9.5 (1:500) 隔夜在4°c。</li>
	<li>
用 PBS 洗掉抗体。
	<ol>
<li>用荧光标记的二级抗体孵化抗原抗体复合体, 室温下孵化30分钟。</li>
		<li>使用一个单独的整除的细胞孵化与二级抗体作为同种控制。在流式细胞术前, 两种细胞在 PBS 中被洗涤和悬浮。</li>
		<li>通过对活细胞进行门控, 分析流式细胞仪中的两个单元群。</li>
	</ol>
</li>
</ol>9. 从 hGFAP 中制备小鼠组织: tdTomato 小鼠
注: 液氧-停止-液-tdTomato cDNA 表达从罗莎的轨迹22,23 (杰克逊实验室, #007914), 并产生内源性荧光当繁殖到鼠标表达的 recombinase (hGFAP)。原位分析是通过生成冰冻组织切片进行的。
<ol><li>将肠道组织固定在4% 多聚甲醛1小时, 然后在 PBS 的1米蔗糖中一夜之间脱水。</li>
	<li>在商业购买的最佳切割组织 (OCT) 化合物中嵌入组织, 由10% 聚乙烯醇和4.2% 聚乙二醇组成, 然后在液氮中凝固。</li>
	<li>准备5µm cryosections, 然后使用带有 DAPI 的 antifade 安装介质安装。</li>
	<li>对于流式细胞术, 准备电梯从 hGFAP: tdTomato + 小鼠, 如步骤4.1–6.5 所述。</li>
	<li>胰蛋白酶处理的细胞从板块后3天的文化和步骤 8.1, 然后修复10分钟4% 多聚甲醛。</li>
	<li>分析从实验性阴性小鼠中分离出来的细胞与从 hGFAP 中分离出来的细胞: tdTomato + 小鼠在流式仪中的作用。 注意: 为了避免死亡的细胞和残骸, 建造了闸门。</li>
</ol>10. Ca2 +通量成像
<ol><li>孵化电梯镀在一个24井板与3µM Fluo-4-AM 37 °c 30 分钟。</li>
	<li>图像的 Ca2 +信号在商业购买 Tyrode 的解决方案使用一个旋转圆盘共焦显微镜和一个激发波长 480 nm (F480) 后加入胆囊收缩素或胃泌肽。对桑德里森等进行了图像分析。10 。</li>
</ol>

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作者没有什么可透露的。

电梯在肠道动态平衡中起着重要作用,在体外分离和研究是十分必要的。在该协议中, 介绍了一种从成年小鼠肠道固有层中分离电梯的简便方法, 研究肠神经胶质功能。
用棉签去除粘附的肠系膜和 LMMP, 可以去除位于纵、圆肌之间的肌间丛胶质细胞, 增加缓冲液对黏膜下表面的可接触性, 并去除大部分大毛细血管.后者减少了红细胞的数量, 污染了最终的文化。本系列 EDTA 孵化剥去上皮粘膜, 露出固有层和黏膜下的胶质细胞, 暴露于螯合溶液后, 可使其脆弱。搅拌要么通过颤抖, 涡流或研制过程 (重复向上和向下吹打) 必须进行仔细和定时, 以避免过度破坏细胞。研磨是在这里使用的, 因为该技术导致了更一致的产量的黏附, 可行的胶质细胞。颤抖倾向于引入气泡并降低细胞的生存能力, 通过细胞粘附到涂层板的形态来评估。组织必须切成块 < 0.5 厘米, 以有效地 triturate 组织与5毫升吸管。
通常, 从至少3只老鼠7厘米段足以产生足够的电梯, 以覆盖一个6井板约20–30% 融合, 然后可以用于免疫细胞化学和 qPCR。然而, 根据所研究的基因的表达水平, 可能需要更多的细胞来进行生化方法, 如西方印迹。虽然, 1 型胶原或胶被简单地研究为替代聚 d-赖氨酸/层粘连蛋白基底, 观察到上皮细胞的更大的黏附, 最终增加胃癌文化的污染与其他细胞类型.此外, 1 型胶原蛋白不牢固地粘在盘子上, 在改变介质时很容易从表面脱落。纤维连接蛋白是另一种已使用24, 但在这里没有测试的基板。层粘连蛋白明显增强了神经嵴衍生细胞的结合和分化25。然而, 许多层粘连蛋白可以改变, 影响细胞黏附和产量。微生物污染的文化发生了大约5% 的时间, 并保持在最低限度的使用无菌试剂, 技术和抗生素。抗菌试剂的使用是可选的。
这项协议的主要局限性是, 在不损害底层电梯的情况下, 将上皮细胞移除的目的是标准化的。此外, 不能立即对制剂进行评估, 因为这取决于细胞是否附着在涂层板上。三要重点解决的领域包括: 1) 使用新鲜的 PDL 和层粘连蛋白来涂板;2) 通过增加助理人数, 将解剖时间缩短至开始 EDTA 孵化;3) 量化用于搅动组织的时间和方法, 有效地去除上皮细胞, 然后电梯。在上述任一步骤中延长时间会增加胃癌的脆弱性, 并且在增长介质中电镀时它们不会恢复。虽然在这里使用了分离上皮的研磨方法, 但震动和涡流也有不一致的结果, 可能是因为它的操作者依赖和难以量化。一旦镀膜后, 如果细胞不被 16–24 h 的干扰, 黏附细胞的产量就会更大。
本文所提出的方法是根据史密斯等的原始研究进行修改的。12, 专注于隔离 LMMP。史密斯的方法是在这里使用, 只是为了去除和丢弃 LMMP, 使大多数的胶质细胞分离的是来自黏膜下层和固有层。此外, 没有酶消化, 肌间丛电梯不容易解放12。然而, 由于肌间丛与黏膜下胶质细胞没有特异的标记, 肌间丛丛中的胶质干细胞的存在是不能排除的。鲍姆细胞分析电梯表达增强型绿色荧光蛋白 (EGFP) 的 hGFAP 促进剂。他们使用非常幼小老鼠 (产后天 7) 和被隔绝的电梯从整个肚腑由酵素消化19。此外, 在维持自由漂浮干细胞形成的两周的条件下, 进行了流细胞分析。虽然作者报告说, neurospheres 高度表达了 GFAP 和 S100b, 浮动细胞聚集也强烈表达α SMA, 表明上皮细胞细胞数量的扩大鼓励 gliosphere 发展的对比到这里描述的扁平板状形态。因此, 虽然有趣的是, 这项研究并不直接与本议定书相媲美。相比之下, 细胞的形态学与明显较少的α SMA + 细胞相结合表明, 本协议所描述的电梯在3至5天的培养期内不表现出3维形态学。然而, 这里的协议和鲍姆方法都使用荧光记者来识别和隔离的 GFAP + 细胞, 这将提高调查员的能力进行活细胞分析。
最后, 本议定书描述了用非酶法从黏膜下层分离肠神经胶质细胞。整个协议约3小时从小鼠解剖到初始电镀的预涂组织培养板。该协议中最耗时的一步是去除和准备第一次 EDTA 孵化的肠道。强烈建议在 DPBS 中准备盘子和储存。该制剂的成功与否取决于有效去除上皮, 而不损害基础电梯和加入 PDL/层粘连蛋白涂层板在24小时内. 初级电梯对体外研究如生化分析有用,Ca2 +通量和腺病毒 transfections10。预计从黏膜下层或 LMMP 分离电梯的能力将允许进一步的研究, 以确定胃癌生长特性, 分化和形态学的差异, 使用全基因组方法。

由于其在肠道完整性和稳态1,2中公认的作用, 对肠道胶质细胞 (电梯) 功能的兴趣稳步增加。另外, 电梯根据他们的位置沿胃肠道3,4的长度变化。电梯释放各种营养因子, 包括胶质细胞源性神经营养因子 (GDNF), 有助于肠道动力1,5 , 并对微生物副产物6,7作出反应。研究表明, 胃癌的种群是异构的, 它们的功能取决于它们是黏膜下的还是驻留在肌间丛丛1,7。例如, 黏膜下层的电梯有助于紧密连接8。电梯中的差异性 GFAP 表达和磷酸化与帕金森氏病有关, 表明它们可能与这种疾病的肠道表型有联系9。最近, 据观察到, 从近端肠道黏膜下层电梯分离培养的核蛋白叶普斯镇的丢失足以诱发激素胃泌素10的表达。因此, 有人建议电梯可能是十二指肠 gastrinomas 的起源, 一种神经内分泌肿瘤10。总的来说, 这些例子突显了研究孤立电梯在神经病理性疾病和癌症11中的行为和功能的相关性。
这一领域的挑战仍然是如何在体外分离和研究胃癌种群。血统痕迹实验表明, 电梯在黏膜下层和固有层的起源于祖细胞在肌间丛丛7。尽管有几种已发布的隔离协议可用于生成肌间丛电梯12、13、14、15、16、17的区域性, 18,19, 没有明确的目标是孤立的黏膜下/叶片固有胃癌人口。现有的胃癌隔离协议专门使用机械分离或平滑肌肉的显微解剖结合酶解分离, 最终丢弃粘膜细胞层。
本手稿的目的是为了证明从固有层的非酶分离初级电梯的步骤,在体外研究。由于没有明确区分肌间丛电梯与黏膜下层的标记, 利用平滑肌上皮黏膜的空间分离来分离黏膜下电梯。此外, 通过将 EDTA 螯合剂与非酶解分离相结合, 电梯从黏膜下层分离出来, 与平滑肌相比较, 与肌间丛电梯。在神经胶质细胞基质上培养细胞,如聚 d-赖氨酸和层粘连蛋白, 进一步分离黏膜下层和固有层电梯。

<table><tbody><tr><td>Poly-D lysine (1 mg/mL stock)</td><td>Sigma</td><td>A-003-E</td><td>Dilute 1:10</td></tr><tr><td>Laminin (0.5 mg/mL stock)</td><td>Sigma</td><td>L4544</td><td>Dilute to 10 &amp;micro;g/mL on ICE</td></tr><tr><td>EDTA (0.5 M)</td><td>Lonza</td><td>51201</td><td>Dilute 1:100 in DPBS</td></tr><tr><td>HEPES (1 M)</td><td>Corning</td><td>36216004</td><td>Dilute 1:100 in DPBS</td></tr><tr><td>Cell Recovery Solution</td><td>Corning</td><td>354253</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>HyClone</td><td>SH30028.02</td><td></td></tr><tr><td>DMEM/F-12</td><td>Thermo Fisher Scientific</td><td>11320033</td><td></td></tr><tr><td>Penicillin-Streptomycin (100x)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Gentamicin (50 mg/mL stock)</td><td>Life Technologies</td><td>15750060</td><td></td></tr><tr><td>GDNF (10 &amp;micro;g stock)</td><td>Sigma</td><td>SRP3200</td><td></td></tr><tr><td>L-Glutamine (200 mM stock)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Chicken anti-GFAP</td><td>Thermo Fisher Scientific</td><td>PA1-10004</td><td></td></tr><tr><td>Goat anti-a-Smooth Muscle Actin&amp;nbsp;</td><td>Abcam</td><td>ab112022</td><td></td></tr><tr><td>Mouse anti-Pgp9.5&amp;nbsp;</td><td>Novus Biologicals</td><td>NB600-1160</td><td></td></tr><tr><td>Goat anti-E-cadherin&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>AF748</td><td></td></tr><tr><td>Rabbit S100&amp;nbsp;</td><td>Abcam</td><td>ab34686</td><td></td></tr><tr><td>Mouse p75 NTR&amp;nbsp;</td><td>Millipore</td><td>MAB5592</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Chicken IgY</td><td>Invitrogen</td><td>A-11039</td><td></td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Goat IgG</td><td>Invitrogen</td><td>A-11055</td><td></td></tr><tr><td>Alexa Fluor 568 Goat Anti-Mouse IgG</td><td>Invitrogen</td><td>A-11004</td><td></td></tr><tr><td>Alexa Fluor 594 Donkey Anti-Rabbit IgG</td><td>Invitrogen</td><td>R-37119</td><td></td></tr><tr><td>Prolong Gold antifade Reagent with DAPI</td><td>Thermo Fisher Scientific</td><td>P36931</td><td></td></tr><tr><td>Fungizone (Amphotericin B) 250 &amp;micro;g/mL</td><td>Life Technologies</td><td>15290-018</td><td></td></tr><tr><td>L-Fura-2-AM</td><td>Invitrogen</td><td>F-14201</td><td></td></tr><tr><td>CCK peptide</td><td>Anaspec, Fremont, CA</td><td>AS-20741</td><td></td></tr><tr><td>Gastrin peptide (Gastrin-17)</td><td>Abbiotec, Bloomington, IN</td><td>350188</td><td></td></tr><tr><td>Nylon Mesh Celll Strainer (100 &amp;micro;m)</td><td>Fisher Scientific</td><td>22363549</td><td></td></tr><tr><td>Nylon Mesh Celll Strainer (40 &amp;micro;m)</td><td>Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>Disposable Serologic Pipet 5 mL</td><td>Fisher Scientific</td><td>13-678-11D</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>25200-056</td><td></td></tr></tbody></table>

isolation of enteric glial cells from the submucosa and lamina propria of the adult mouse

肠道神经系统 (电梯) 由位于平滑肌壁、黏膜下层和固有层内的神经细胞和肠胶质细胞组成。电梯在肠道动态平衡中发挥重要作用, 通过释放各种营养因素, 促进上皮屏障的完整性。大多数对原发性肠神经胶质细胞的研究都是在酶解离肌间丛丛中分离出来的。本文介绍了一种非酶法分离和培养电梯肠黏膜下层和固有层的方法。在人工清除纵向肌肉层后, 电梯从固有膜和黏膜下层解放出来, 使用顺序 HEPES 缓冲 EDTA 孵化, 然后在商业上可用的非酶细胞回收解决方案孵化。EDTA 孵化足够从固有层剥离大部分上皮粘膜, 使细胞恢复解决方案解放黏膜下电梯。任何残余的固有层和平滑的肌肉被丢弃连同肌间丛胶质细胞。电梯很容易通过表达胶质纤维酸性蛋白 (GFAP) 的能力来鉴别。只有约50% 的细胞悬浮液中含有 GFAP + 细胞完成组织孵化后, 在电镀之前, 聚 d-赖氨酸/层粘连蛋白基底。然而, 经过3天的培养细胞的神经胶质细胞源性神经营养因子 (GDNF) 包含培养基, 细胞的数量附着在基板涂层板组成的 > 95% 肠道胶质。我们创建了一个混合的鼠标线, 通过培育 hGFAP 鼠到罗莎-tdTomato 的记者线, 以跟踪的百分比的 GFAP + 细胞使用内源性细胞荧光。因此, 非肌间丛肠胶质细胞可通过非酶法分离, 培养至少5天。

眼影被认为是不成功的, 如果 GFAP + 细胞没有坚持和传播24小时内 (图 4A)。在24小时后, 当细胞粘附并显示有扩散成扁平骨料的证据时, 胶质细胞的数量无法确定 (图 4B)。星团边缘的细胞倾向于延伸长时间的过程, 并表达了经典的胶质标记,例如, GFAP, S100b 和 p75NTR (图 4C, 4D)10,16。到了 2天, 胶质细胞紧紧地附着在表面上, 使非黏附的人群可以用 PBS 冲洗掉。大多数漂浮细胞都是上皮细胞和固有细胞膜, 以及细胞碎片。这些漂浮细胞团簇的存在降低了粘附胶质细胞的产量, 强调了孵化 EDTA 的重要性, 直到流经几乎清楚, 信号表明上皮细胞已从叶片中移除。固有的核心。此外, 在细胞恢复解决方案孵化后, 速度低于 1500 x g的离心, 不能有效地将所有可行的细胞收集到导致产量下降的颗粒中。典型地, 4万到10万细胞形态上一致与电梯坚持在3天在文化。
免疫组化分析与胶质相关的抗体, 表明在3天保持黏附分子的细胞簇是 GFAP, S100b 和 Sox 10 阳性10,16 (图 5A)。在目前的研究中, 通过 permeabilizing 细胞和流式细胞术 (图 5B-F) 进行分析, 观察到相同的纯度。流式细胞术在隔离黏膜下/叶片固有神经胶质细胞后立即发现约51% 的细胞是 gfap + (图 5B), 提示存在的胶质细胞类型,如上皮, 造血,内皮细胞, 神经细胞, 肌已知存在于上皮和固有层。在培养3天后, 在去除原始介质后, 用 PBS 轻轻冲洗, 然后添加新鲜的介质 (图 5C), 在细胞总数95% 以上的 GFAP + 细胞数量上进行分析。有趣的是, 流量分析发现高和低的 GFAP 蛋白表达的人群 (图 5C)。事实上, 对细胞的免疫荧光分析显示, 星团的外围倾向于表达更高水平的 GFAP (图 4C, 4D)。两者相结合, 这两种分析可能表明胃癌成熟或分化状态的差异。总的来说, α SMA + (上皮细胞细胞标记), e-钙黏蛋白 + (上皮细胞标记) 和 Pgp 9.5 + (神经元细胞标记) 细胞的百分比小于 5% (图 5D-F)。这些结果与先前的研究一致, 使用免疫荧光标记电梯显示约 93% GFAP + 细胞10。流动分析强调了允许细胞附着在盘子上的重要性, 因为它允许清除包括大约5% 种文化的污染细胞种群。
本研究的目标是使用一个 GFAP 激活的记者, 以促进细胞流式细胞术的进一步分析和比较的程度胃癌浓缩使用内源荧光标记 (图 6)。hGFAP 的鼠标线最初由混乱和工友描述20。简而言之, recombinase 被置于 2.2 kb 的人胶质纤维酸蛋白 (hGFAP) 启动子的控制之下。因此, 任何转录这个 hGFAP 启动子的细胞表达了红色荧光记者 tdTomato。在执行上述胃癌隔离程序之前, 用 tdTomato 报告员标记的细胞群使用肠道和结肠的冰冻切片 (图 6A, 6B) 进行原位可视化。在执行 EDTA/细胞恢复隔离和培养细胞3天后, 这些小鼠的电梯很容易被他们的内源性荧光识别 (图 6C)。虽然可以说, 在近端肠道4,21, tdTomato + 电梯也被观察到结肠 (图 6B) 中的 GFAP + 细胞更大量。使用 hGFAP 激活的 tdTomato+荧光记者也发现了 hGFAP-tdTomato + 细胞的双峰种群 (图 6D), 观察使用了 GFAP 蛋白的免疫荧光分析 (图 5C)。虽然据报道, 并非所有电梯表达的 gfap 蛋白4, 这一点可能反映的差异, 在 gfap 表达的水平。平均来看, 大约66% 的细胞被分析显示 tdTomato 荧光的最高水平。因此, EDTA/细胞恢复协议可以用来隔离电梯的亚群在整个小肠和结肠标记的记者, 以检查差异的基因表达。
该协议产生了足够数量的相对纯净的 GFAP + 胶质细胞的生物化学研究和西方印迹分析10. 为了证明胶质细胞的反应能力, 3 天的胶质细胞准备用激素胆囊收缩素 (胆囊) 或胃胃液来诱导钙2 +通量 (图 7A-7B)10。结果表明, 这些 GFAP + 黏附细胞的反应, 细胞外激动剂表明他们的能力, 显示已知的肠道胶质功能。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57629/57629fig1.jpg"> 图 1: 建立和制备小鼠肠道.(A) 在冰上设置的隔离图, 包括提取的肠道。(B) 附在20克钝端针上的5毫升注射器的图片, 以冲洗粪便内容。(C) 在 DPBS 缓冲区浸泡的木制棉签 (〜4厘米) 的设计结束。<a href="https://www.jove.com/files/ftp_upload/57629/57629fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57629/57629fig2.jpg"> 图 2: 用于清洁肠道和去除纵肌/肌间丛丛 (LMMP) 的步骤.(丙)把肠段滑到一根湿透的木棍上。(D) 切除肠系膜粘连。(E) 用剃刀刀片偷肠道。(F) 用湿棉签除去 LMMP。<a href="https://www.jove.com/files/ftp_upload/57629/57629fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57629/57629fig3.jpg"> 图 3: EDTA 孵化后的连续流动.显示的例子, 从4顺序孵化使用三7厘米小肠段孵化10分钟与5毫米 EDTA/10mM HEPES 在 DPBS, 然后 triturated 20 倍。展示了通过100µm 尼龙网过滤器浇注后的代表性流量。<a href="https://www.jove.com/files/ftp_upload/57629/57629fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57629/57629fig4.jpg"> 图 4: 电镀后的胃癌图像24小时.电镀后悬浮细胞24小时的光镜检查。(A) 显示的是一个有代表性的例子, 显示漂浮上皮细胞团簇 (箭头) 和碎片的不良准备。24小时后没有观察到黏附电梯(B) 显示的是一个典型的例子, 在细胞隔离和泥沙后, 电梯贴在 PDL/层粘连蛋白涂层板上的最佳准备24小时。图像是用数码相机在倒置的荧光显微镜上捕捉到的。刻度条 = 500 µm (A B)。 (C) 高功率视图的胃癌细胞斑块染色的 GFAP 抗体 (绿色) 显示细胞在外围具有较高的标签强度。几个细胞显示双核 (箭头, DAPI, 蓝色)。(D) 定位 (绿色) 与 S100 (红色) 或 p75NTR (红色)。刻度条 = 20 µm (C D)。细胞被安装与 antifade 试剂和 DAPI (蓝色原子核)。<a href="https://www.jove.com/files/ftp_upload/57629/57629fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57629/57629fig5.jpg"> 图 5: 从成年老鼠的固有的十二指肠层中分离出的肠神经胶质细胞流式细胞术.(A) 3 天后, 对胃癌文化的胶质、S100B 和 Sox10 进行免疫荧光。箭头表示 Sox 10 正核。刻度条 = 20 µm (在桑德里森等的允许下使用。10). (B) 涂覆板 (荧光强度 (或正散射、X 轴) 与侧向散射 (Y 轴) 电镀前的 GFAP 阳性细胞百分比。(C) GFAP 阳性 (D) α SMA 阳性 (e) e-钙黏蛋白阳性 (F) 和 Pgp 9.5 阳性细胞 3 daysafter 电镀的百分比。盖茨是为了避免死亡的细胞和残骸而建造的。显示的是荧光填充相对于细胞数的平均百分比 (侧散点)。<a href="https://www.jove.com/files/ftp_upload/57629/57629fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57629/57629fig6.jpg"> 图 6: hGFAP 的十二指肠层固有的肠神经胶质细胞的鉴定+: tdTomato+成年鼠.内源性 tdTomato 荧光 (红色) 图像被捕获的相位对比荧光显微镜与数码相机在 (a) 肠道。嵌入与 DAPI 图像 (蓝色原子核) 合并的除外。(B) 与 DAPI 合并的结肠。(C) 从 hGFAP 中分离出的肠胶质细胞 (红色): dtTomato+小鼠, 在 PDL/层粘连蛋白基底上培养3天。刻度条 = 50 µm (D) tdTomato 细胞的流细胞分析。显示的是平均% 的 GFAP + 细胞的 SEM 为3种不同的准备 (3 只老鼠每准备)。<a href="https://www.jove.com/files/ftp_upload/57629/57629fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57629/57629fig7.jpg"> 图 7: 激素治疗后的 Ca2 +通量的视频。在24井的 PDL 和层粘连蛋白涂层板的电梯镀在胶质生长培养基中培养了3天。细胞预先加载了3µM Fura-2-AM 20 分钟, 37 摄氏度, 然后用 (A) 100 nM 胆囊收缩素或 (B) 100 nM 胃液治疗。<a href="https://www.jove.com/files/ftp_upload/57629/Figure_7.zip">请单击此处下载此文件.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>要添加的卷</td>
			<td>最终浓度</td>
		</tr>
<tr>
<td>DMEM/F12</td>
			<td>44.5 毫升</td>
			<td>_</td>
		</tr>
<tr>
<td>胎牛血清 (血清)</td>
			<td>5毫升</td>
			<td>10%</td>
		</tr>
<tr>
<td>青霉素-链霉素</td>
			<td>500µL</td>
			<td>100个/毫升笔</td>
		</tr>
<tr>
<td>链霉素</td>
			<td></td>
			<td>100µg/毫升链球菌</td>
		</tr>
<tr>
<td>庆大霉素 (50 毫克/毫升)</td>
			<td>20µL</td>
			<td>20µg/毫升</td>
		</tr>
</tbody></table>表 1: 胶质细胞泥沙培养基的组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>要添加的卷</td>
			<td>最终浓度</td>
		</tr>
<tr>
<td>DMEM/F12</td>
			<td>44毫升</td>
			<td>_</td>
		</tr>
<tr>
<td>胎牛血清 (血清)</td>
			<td>5毫升</td>
			<td>10%</td>
		</tr>
<tr>
<td>青霉素-链霉素 (100x)</td>
			<td>500µL</td>
			<td>100个/毫升 (笔)</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>100µg/毫升 (链球菌)</td>
		</tr>
<tr>
<td>庆大霉素 (50 毫克/毫升)</td>
			<td>20µL</td>
			<td>20µg/毫升</td>
		</tr>
<tr>
<td>GDNF (10 µg/毫升股票)</td>
			<td>50µL</td>
			<td>10 ng/毫升</td>
		</tr>
<tr>
<td>l-谷氨酰胺 (200 毫米库存)</td>
			<td>500µL</td>
			<td>2毫米</td>
		</tr>
</tbody></table>表 2: 胶质细胞生长培养基的组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>免疫荧光稀释</td>
			<td>流式细胞仪稀释</td>
		</tr>
<tr>
<td>鸡抗胶质蛋白</td>
			<td>1到500</td>
			<td>1到2000</td>
		</tr>
<tr>
<td>兔 anti-S100</td>
			<td>1到500</td>
			<td>1到500</td>
		</tr>
<tr>
<td>鼠标 anti-p75 NTR</td>
			<td>1到500</td>
			<td>1到500</td>
		</tr>
<tr>
<td>山羊抗 e-钙黏蛋白</td>
			<td></td>
			<td>1到400</td>
		</tr>
<tr>
<td>鼠标抗 Pgp9。5</td>
			<td></td>
			<td>1到500</td>
		</tr>
<tr>
<td>山羊抗平滑肌肌动蛋白</td>
			<td></td>
			<td>1到500</td>
		</tr>
<tr>
<td>488山羊抗鸡 IgY</td>
			<td>1到1000</td>
			<td>1到1000</td>
		</tr>
<tr>
<td>568山羊抗鼠 IgG</td>
			<td>1到1000</td>
			<td>1到1000</td>
		</tr>
<tr>
<td>594驴抗兔 IgG</td>
			<td>1到1000</td>
			<td></td>
		</tr>
<tr>
<td>488驴抗山羊 IgG</td>
			<td></td>
			<td>1到1000</td>
		</tr>
</tbody></table>表 3: 抗体列表

Watch this Scientific Journal Video about Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse at JoVE.com

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

在这里, 我们描述了肠道黏膜下层的肠神经胶质细胞的分离, 使用顺序 EDTA 孵化螯合价阳离子, 然后孵化在非酶细胞恢复解决方案。在聚 d-赖氨酸和层粘连蛋白的作用下, 电镀细胞悬浮物会导致黏膜下胶质细胞的高度富集培养, 用于功能分析。

成年小鼠黏膜下层和固有层肠道胶质细胞的分离

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

isolation-enteric-glial-cells-from-submucosa-lamina-propria-adult

Nanyang Technological University

Research

JoVE Journal

Biology

13.0K 次观看.  University of Michigan. 在这里, 我们描述了肠道黏膜下层的肠神经胶质细胞的分离, 使用顺序 EDTA 孵化螯合价阳离子, 然后孵化在非酶细胞恢复解决方案。在聚 d-赖氨酸和层粘连蛋白的作用下, 电镀细胞悬浮物会导致黏膜下胶质细胞的高度富集培养, 用于功能分析。

视频: Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

科研

免疫与感染

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

神经科学

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

生物学

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

从成年小鼠脊髓中高效提取星形胶质细胞和小胶质细胞

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

作者聚焦:从成年小鼠脊髓中分离神经胶质细胞 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成年小鼠泪液和下颌腺肌上皮细胞的分离

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

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