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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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glial+cells+and+their+role+in+the+intestinal+epithelial+barrier.">Enteric glial cells and their role in the intestinal epithelial barrier.</a> World Journal of Gastroenterology. 20 (32), 11273-11280 (2014).</li><li>Clairembault, T., 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=Enteric+GFAP+expression+and+phosphorylation+in+Parkinson's+disease.">Enteric GFAP expression and phosphorylation in Parkinson's disease.</a> Journal of Neurochemistry. 130 (6), 805-815 (2014).</li><li>Sundaresan, 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=Gastrin+Induces+Nuclear+Export+and+Proteasome+Degradation+of+Menin+in+Enteric+Glial+Cells.">Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.</a> Gastroenterology. 153, 1555-1567 (2017).</li><li>Gulbransen, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+Glia:+The+Origin+of+Duodenal+Gastrinomas?.">Enteric Glia: The Origin of Duodenal Gastrinomas?.</a> Gastroenterology. , (2017).</li><li>Smith, T. H., Ngwainmbi, J., Grider, J. R., Dewey, W. L., Akbarali, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+preparation+of+isolated+enteric+neurons+and+glia+from+the+myenteric+plexus+of+the+adult+mouse.">An in vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Fried, D. E., Gulbransen, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+Ca2++imaging+of+the+enteric+nervous+system.">In situ Ca2+ imaging of the enteric nervous system.</a> Journal of Visualized Experiments. (95), (2015).</li><li>Bernstein, C. N., Vidrich, 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,+identification,+and+culture+of+normal+mouse+colonic+glia.">Isolation, identification, and culture of normal mouse colonic glia.</a> Glia. 12 (2), 108-116 (1994).</li><li>Jaeger, C. 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B., 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=Distribution+of+enteric+glia+and+GDNF+during+gut+inflammation.">Distribution of enteric glia and GDNF during gut inflammation.</a> BMC Gastroenterology. 11, 3 (2011).</li><li>Rosenbaum, C., 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=Activation+of+Myenteric+Glia+during+Acute+Inflammation+In+Vitro+and+In+Vivo.">Activation of Myenteric Glia during Acute Inflammation In Vitro and In Vivo.</a> Public Library of Science One. 11 (3), e0151335 (2016).</li><li>Zhuo, L., 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=hGFAP-cre+transgenic+mice+for+manipulation+of+glial+and+neuronal+function+in+vivo.">hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo.</a> Genesis. 31 (2), 85-94 (2001).</li><li>Boesmans, W., Lasrado, R., Vanden Berghe, P., Pachnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+and+phenotypic+plasticity+of+glial+cells+in+the+mammalian+enteric+nervous+system.">Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system.</a> Glia. 63 (2), 229-241 (2015).</li><li>Shaner, N. C., 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=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nature Biotechnology. 22 (12), 1567-1572 (2004).</li><li>Madisen, L., 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=A+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain.">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nature Neuroscience. 13 (1), 133-140 (2010).</li><li>Kabouridis, P. 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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?...

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 &#181;g/mL on ICE</td>
		</tr>
		<tr>
			<td>EDTA (0.5M)</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&#39;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 (50mg/mL stock)</td>
			<td>Life Technologies</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF (10 &#181;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&#160;</td>
			<td>Abcam</td>
			<td>ab112022</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Pgp9.5&#160;</td>
			<td>Novus Biologicals</td>
			<td>NB600-1160</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-E-cadherin&#160;</td>
			<td>R&#38;D Systems</td>
			<td>AF748</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit S100&#160;</td>
			<td>Abcam</td>
			<td>ab34686</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse p75 NTR&#160;</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 &#181;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 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Celll Strainer (40 &#181;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)<sup class="xr...

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

Research

JoVE Journal

Biology

12.9K 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 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 ...

科研

生物学

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

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, ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...