我们感谢芭芭拉Carletti和安娜玛丽肯尼宝贵的意见。 HYL是支持的培训津贴“荷尔蒙：生物化学与分子生物学”，DK07328。支持由美国国立卫生研究院授予5R01 NS16951（CAM）的一部分。

第1部分：设置（清扫术前1-2天）（不显示视频） <ol><li>编制文化的解决方案和介质： <ul><li> 4X CMF - PBS - EDTA（钙和镁的磷酸盐缓冲液EDTA Percoll稀释）：每公升，加入32 g氯化钠，氯化钾1.2克，8克葡萄糖，2克的NaH 2 PO 4，1克KH 2 PO 4，8毫升2％碳酸氢钠3股票，10毫升1M EDTA（pH 8.0）的蒸馏水/去离子水。调节音量到1升，pH值至7.4。过滤消毒。 </li><li> HBSS中葡萄糖：加入葡萄糖（6克/升），钙和镁的自由汉克的平衡盐溶液（HBSS）和无菌过滤。在4 ° C为一个月，可存储500毫升。 </li><li>文化传媒：无血清培养基（SFM），和10％胎牛血清的介质。 48毫升的Neurobasal一个中等加500μL100X GlutaMAX我，500μL100X青霉素，链霉素（最后100个单位的青霉素和100微克链霉素），6.25μL2米氯化钾（最终250微米）。分割成2等份9毫升和40毫升。要准备10％胎牛血清培养基，添加1ml至9毫升分装热灭活的FB。要准备的SFM，添加800μL无血清的补充B - 27到40毫升等分。过滤消毒和储存在4 ° C，最高为2周。为了达到最佳效果，使每个实验的新媒体。 </li></ul></li><li>编制PERCOLL稀释： <ul><li> Percoll是提供一种液体，使用前必须先酸化。要准备的股票，加入1N盐酸很少很少超过1-2小时内Percoll解决，直到pH值达到7.4。添加盐酸过快，会导致总Percoll。大约1N盐酸6.5毫升需要为每公升的Percoll。过滤消毒，并储存于4 ° C。 </li><li> Percoll稀释了35％和60％（卷：第一卷）将用于在Percoll梯度步骤。准备100毫升Percoll稀释含有35或60毫升Percoll股票，4X CMF - PBS - EDTA和蒸馏水25毫升/去离子水到终体积。添加几滴甲苯胺蓝色60％的解决方案，这将有助于可视化界面和细胞。过滤消毒，并储存于4 ° C。 </li><li> 4X CMF - PBS - EDTA不应超过3个月以上，不足缓冲在操作过程中会造成细胞死亡的增加。 </li></ul></li><li>准备盖玻片：盖玻片（最好的，如果从北卡罗来纳州生物供应公司）洗净，用10％盐酸在室温下用温和的搅拌过夜，蒸馏水/ deonized水彻底冲洗，并存储在70％的乙醇。在使用之前，单盖玻片火焰消毒简要明火，直到乙醇蒸发（本生灯）用镊子。 4孔培养板和25​​毫米盖玻片，12毫米的玻璃盖玻片，可放置在6孔培养板。 </li><li>涂层与基体培养容器：对于免疫荧光染色或药物治疗盖玻片，是比较合适的，因为它们可以轻松地安装在载玻片上，在显微镜下可视化。当收集蛋白质或RNA样品需要大量的细胞，细胞培养塑料培养dishes.The晚上之前清扫，盖玻片或塑料的文化菜肴必须涂上聚- D -赖氨酸（500微克/毫升; 800μL/ 6孔板或350μL/ 4孔板）。储存在-20 ° C，然后稀释后，在无菌蒸馏水/ deonized水和无菌过滤前使用权，5毫克/毫升原液聚- D -赖氨酸。培养容器培养，在37 ° C过夜（或至少2小时前电镀）和两次电镀前洗净，用无菌蒸馏水/ deonized水权。 </li><li>准备PREPLATING菜：这些菜将被用于镀前孤立的小脑细胞培养前去除胶质污染物，更容易坚持到低浓度的基板。大衣两个60毫米的4毫升每盘聚- D -赖氨酸（请注意，这是培养使用浓度的1 / 5，100微克/毫升）的塑料培养皿。在37 ° C孵育过夜（或至少2小时前清扫）。解剖前，删除D -聚赖氨酸的解决方案，用无菌水冲洗两次菜，让干在引擎盖。 </li><li>消毒的高压灭菌器或一天清扫的清扫工具，沉浸在70％乙醇20分钟的工具。所需工具：Permaset剪刀，microdissecting剪刀，四个杜蒙＃5解剖钳。 </li></ol>第2部分：准备夹层（夹层一天） - （视频演示） 所有下列程序进行，除非指出在组织培养罩。 <ol><li>用70％乙醇擦拭解剖区域。媒体预热至37 ° C的水洗澡或5％的合作2 37 ° C培养箱。 </li><li>当开始一个新的木瓜蛋白酶分解系统套件（顿），准备白蛋白，卵类粘蛋白抑制剂的解决方案。添加32毫升EBSS（Earle的平衡盐溶液;工具包中的提供），白蛋白，卵类粘蛋白抑制剂的混合物和允许的内容，以解散而准备的其他组成部分。第一次使用的重组，然后将其存储其余的解决方案，在2-8 ° C和使用，直至完成每个套件的五个允许的隔离。 </li><li>从离解包（每小瓶足够的解离4日至15日从产后第5天小鼠的小脑）的木瓜蛋白酶小瓶添加5毫升的EBSS。木瓜小瓶放置在5％CO 2 37 ° C培养箱或37 ° C为5至10分钟的水浴，直至木瓜蛋白酶是完全溶解和明确的解决方案出现。在室温保持在清扫的解决方案。 </li><li>加入500μLEBSS一个DNA酶小瓶从离解包。轻轻拍打小瓶因为DNA酶敏感剪切变性混合。这个解决方案中加入250μL含有木瓜蛋白酶（终浓度为20单位/毫升的木瓜蛋白酶和0.005％DNA酶）的小瓶。在步骤5.1或6.1中使用的保存剩余的DNA酶小瓶。 </li></ol>第3部分：夹层和脑膜去除<ol><li> C57BL/6J小鼠颗粒细胞培养的最佳年龄是产后4-6天，当颗粒细胞祖细胞数量在EGL peaks1，10。解剖幼仔一次，你变得更加熟悉与小脑剥离，尽量减少清扫时间不超过6分钟，每小狗。速度是本质。 如果Percoll梯度步骤旁路，八天产后5鼠标幼仔可以产生足够的细胞，一个6孔培养板（或6个25毫米盖玻片）或6个4孔培养板（或二十四个月12日 - 毫米盖玻片）。近似​​的产量是每4至5x106细胞小狗和镀层的密度范围可介于1和5X105 cells/cm2。由于15-20％的细胞在Percoll STEP4丢失，需要更多的动物，以获得所需的细胞密度。 </li><li>移液器的HBSS葡萄糖15毫升到60毫米的塑料组织培养皿和10毫升到无菌的猎鹰15 ml锥形塑料管。置于冰上。 </li><li>罩外，擦拭用70％乙醇的小狗的头。使用Permaset剪刀，杀头的小狗。 （斩首不会显示在视频。） </li><li>在引擎盖上，与杜蒙钳头，让您可以清楚地看到后面的头骨。进入枕骨大孔插入microdissecting剪刀，减少对眼睛直的大脑。使用镊子，剥离皮肤，抬起头颅大脑暴露。杜蒙钳，夹断小脑和周围脑转移到菜的HBSS葡萄糖。 （这是比较容易操纵的小脑及删除脑膜，小脑仍连接到周围组织）。 </li><li>解剖显微镜下，轻轻剥离关闭小脑脑膜之间也有一个优良杜蒙＃5，而使用其他镊子钳，锚定板小脑的叶。你会发现小脑表面血管。这些血管是一个很好的的方法来识别脑膜。取出的脑膜，直到小脑上乱蓬蓬的白色外观。从脑的其余部分隔开的小脑。把它转化为腹侧和删除的脉络丛，看起来像一个红色的缎带之间的腹侧小脑和相邻的脑。放置在一个15毫升的锥形管的冰尽快解剖每个小脑冷的HBSS -葡萄糖。更改新鲜的HBSS葡萄糖解剖3大脑解剖后的解决方案。 </li></ol>第4部分：细胞悬液<ol><li>一旦完成所有的解剖，从管中删除的HBSS -葡萄糖和步骤2.4木瓜蛋白酶溶液取代。虽然该工具包建议切小块组织，我们发现，这是没有必要削减或剁碎在这个年龄段的小脑。将组织+木瓜蛋白酶溶液（在15毫升的锥形管）在37℃水浴，或5％的CO 2 37 ° C培养箱中培养15至20分钟。 4日至8小脑，15分钟，在37 ° C是足够的; 20至30分钟为一个较大的小脑是更好。轻轻摇动试管，每3至4分钟。 </li><li>磨碎用无菌的P1000气溶胶枪头已与胎牛血清涂混合物。做到这一步，轻轻地，以防止任何气泡的形成。消防抛光玻璃移液器可以使用，但我们发现，血清涂无菌P1000气溶胶提示很好的工作。大衣与FBS枪头，吸管FBS的向上和向下使用前的两倍。用移液管约10上下运动，有足够的游离组织。该解决方案将变得浑浊。允许暂停坐30-60秒，未解离组织的任何大块将落户试管底部。 </li><li>取出悬浮细胞（小心不要删除未解离的组织块，在Bottom）进入一个新的15毫升血清涂枪头锥形管。离心5分钟，在室温约200xg。准备再悬浮液。要做到这一点，混合重组白蛋白，卵类粘蛋白抑制剂在无菌试管溶液与300μL2.7毫升EBSS。从步骤2.4中添加150μL的DNA酶的解决方案。 </li><li>离心后，弃去上清液，并立即重悬细胞沉淀在稀释的DNA酶/白蛋白抑制剂的解决方案，在步骤4.3准备与血清涂层P1000气溶胶枪头。 </li><li>准备如下一个连续密度梯度。添加5毫升，一个15毫升的锥形管的白蛋白，卵类粘蛋白抑制剂的解决方案。仔细层细胞悬液，在上面。在约70xg离心机在室温下6分钟。游离细胞在试管底部沉淀，膜碎片留在接口。 </li><li>如果你想获得颗粒细胞丰富的文化，绕过Percoll梯度分离步骤（产量小脑颗粒细胞的素净文化），并进行第6部分。如果你想进一步孤立Percoll梯度分离的神经胶质细胞和大interneurons的颗粒细胞，进行到第5。 Percoll梯度步骤的使用将减少细胞产量。在我们的手中，在产量减少约20-35％。然而，从约90％到95-99％浓缩颗粒神经元细胞和祖细胞的增加。 </li></ol>第5部分：Percoll梯度分离<ol><li>弃上清，并立即重悬在2毫升的HBSS葡萄糖颗粒与脱氧核糖核酸酶50μL步骤2.4。虽然尼龙网（孔径70微米）通过重力过滤细胞悬液。这一步将删除大非神经元细胞，并提供了一​​个更好的单细胞悬液。 </li><li>准备两个火抛光的玻璃巴氏吸管。要做到这一点，一个玻璃吸管轻轻火焰尖端，直至其边缘是理顺和吸管孔是原始大小的40-50％。小心不要使开放太小。 </li><li>准备Percoll梯度。将10毫升35％在50毫升的无菌锥形管Percoll溶液。 60％Percoll（10毫升），装入10毫升的无菌注射器，用无菌脊髓针连接。轻轻层的60％，35％的解决方案下面Percoll溶液，加入60％的解决方案与脊髓针管的底部。小心，以保持锋利的两层之​​间的接口。细胞加入到使用火抛光的吸管的梯度的顶部，轻轻沿管的一侧，而不会干扰接口。 </li><li>小心地在1800xg的离心机和离心机的管。斜坡的速度一步每20秒（速度递增如下：约18，70，200，440，850，1100，1800xg），所以它大约需要2分钟，以达到所需的速度。达到1800xg时开始的10分钟定时器。结束时，下降速度逐渐一步，每20秒。 </li><li>删除渐变的上部接口（含大量的神经胶质细胞，浦肯野细胞，并interneurons）并丢弃。 </li><li>小心取出的35％和60％Percoll之间的接口与火抛光的吸管，并转移至50 ml锥形管的细胞。悬浮在3卷HBSS中葡萄糖和反相几次混合。 1100xg离心5分钟。 </li><li>去除上清，添加4毫升10％胎牛血清的介质中，用50μL的DNA酶。悬浮颗粒轻轻地使用火抛光的吸管，形成单细胞悬液。继续到步骤6.2。 </li></ol>第6部分：预镀和电镀<ol><li>弃上清，并立即在4毫升10％胎牛血清培养基重悬沉淀与脱氧核糖核酸酶50μL步骤2.4。虽然尼龙网（孔径70微米）通过重力过滤细胞悬液。这一步将删除大量的非神经元细胞，并提供了一​​个更好的单细胞悬液。 </li><li>板块上一个D -聚赖氨酸涂层镀前20分钟的菜在5％CO 2 37 °彗星孵化器收集细胞。重复同一个新鲜的菜。星形胶质细胞和较重的细胞会安顿下来，并坚持的菜肴和留下的小颗粒神经元和神经元祖细胞的浮动。坚持松散的颗粒神经元和神经元祖细胞的潜伏期后，很容易脱落，并与温柔攻板松动。 </li><li>在200xg 5分钟，收集在15毫升锥形管和离心颗粒神经元和神经元祖细胞。 1毫升的无血清培养基重悬的颗粒，并使用一个血球计数分装10μL。 </li><li>加入无血清培养基，以达到所需的细胞密度。粗糙的指引，以板中密度细胞的数量：6孔板，350万至4万细胞; 4孔板，65细胞。我对于6孔培养板，板1.5毫升/ 4 - PLA工商业污水附加费，我板0.5毫升/孔。细胞保持在5％CO 2 37 °彗星孵化器。彻底改变中期24小时后每两到三天以后的变化。 </li></ol>

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	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of the cerebellar system: in relation to its evolution, structure, and functions. , (1997).</li><li>Hatten, M. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Annual Review of Neuroscience. 18, 385-285 (1995).</li><li>Hatten, M. E., Gao, W. -. Q., Morrison, M. E., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in Culturing Nerve Cells. , 419-419 (1998).</li><li>Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of Cell Biology. 100 (2), 384-384 (1985).</li><li>Carletti, B., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroscientist. 14 (1), 91-91 (2008).</li><li>Knoepfler, P. S., Kenney, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Cycle. 5 (1), 47-47 (2006).</li><li>Manzini, M. C., Joseph, D. J., MacDermott, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular and Cellular Neuroscience. 35 (2), 328-328 (2007).</li><li>Manzini, M. C., Ward, M. S., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Journal of Neuroscience. 26 (22), 6040-6040 (2006).</li><li>Baptista, C. A., Hatten, M. E., Blazeski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 12 (2), 243-243 (1994).</li><li>Palay, S. L., Chan-Palay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cerebellar Cortex: Cytology and Organization. , (1974).</li><li>Messer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brain Research. 130 (1), 1-1 (1977).</li><li>Ruiz I Altaba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development. 126, 3205-3205 (1999).</li><li>Wallace, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Biology. 9 (8), 445-445 (1999).</li><li>Wechsler-Reya, R. J., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 22 (1), 103-103 (1999).</li><li>Solecki, D. J., Liu, X. L., Tomoda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 31 (4), 557-557 (2001).</li><li>Powell, S. K., Williams, C. C., Nomizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Journal of Neuroscience Research. 54 (2), 233-233 (1998).</li><li>Lander, A. D., Fujii, D. K., Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (7), 2183-2183 (1985).</li><li>Kenney, A. M., Rowitch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular and Cellular Biology. 20 (23), 9055-9055 (2000).</li><li>Kenney, A. M., Cole, M. D., Rowitch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development. 130 (1), 15-15 (2003).</li><li>Pons, S., Trejo, J. L., Martinez-Morales, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development. 128, 1481-1481 (2001).</li></ol>

这个协议是基于已在过去的3,7,11描述程序的修改。有几个重要的点要注意下面讨论。 坚持在2小时电镀内的颗粒神经元细胞和祖细胞。健康的细胞有在相衬显微镜 4下一轮形态。电镀后的24小时之内，健康的细胞将盖玻片或塑料以及周围均匀分布，并会形成过程。第一介质的变化时，24小时后，将有几个死浮动细胞。这是正常的，少数细胞会死亡或解离过程中变得不健康。然而，如果48小时后?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell strainer with 70&mu;m mesh pore</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>Spinal Needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>405182</td>
<td>20G x 3-1/2 inches</td>
</tr>
<tr>
<td>Poly-D-Lysine mol wt>300,000</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1024</td>
<td>Each new batch of Poly-D-Lysine must be tested. 
</td>
</tr>
<tr>
<td>Percoll</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-1644</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin (100X)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4333</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175-103</td>
<td>Must be calcium and magnesium free.</td>
</tr>
<tr>
<td>Neurobasal A-Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10888-022</td>
<td>&nbsp;</td>
<tr>
<td>Glutamax I supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum Free Supplement (50x)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>12-mm circle coverslips </td>
<td>&nbsp;</td>
<td>Carolina Biological Supply</td>
<td>63-3029</td>
<td>These are made in Germany by Deckgluder.</td>
</tr>
<tr>
<td>25-mm circle coverslips</td>
<td>&nbsp;</td>
<td>Carolina Biological Supply</td>
<td>63-3037</td>
<td>These are made in Germany by Deckgluder.</td>
</tr>
<tr>
<td>Papain Dissociation System </td>
<td>&nbsp;</td>
<td>Worthington Biochemical Corporation</td>
<td>LK 003150</td>
<td>Kit is good for five isolations.</td>
</tr>
<tr>
<td>Permaset scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS6782</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 (Dumostar)</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS4978</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture dish</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture dish</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140685</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons 

小脑皮层是一个很好的描述的结构，为研究神经元的性质和发展1,2提供了独特的机会。小脑的神经元类型（颗粒细胞，浦肯野细胞和抑制interneurons），颗粒神经元是目前最众多的和最丰富的哺乳动物的大脑中的神经元类型。在啮齿类动物中，小脑颗粒神经元在小脑皮质，外部颗粒层（EGL）的最外层产后的前两个星期祖细胞生成的。这里介绍的协议描述的技术来丰富和文化产后小鼠小脑颗粒神经元祖细胞。我们将描述的程序，以获取增加纯度 3,4 文化，它可以被用来研究颗粒神经元的增殖到5,6祖细胞的分化。一旦祖细胞分化，文化也提供实验操作和现象，如突触谷氨酸受体功能 7，与其他纯化的小脑细胞8,9或细胞死亡7的互动特征的颗粒神经元的同质人口。

Watch this Scientific Journal Video about Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons at JoVE.com

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

在这里，我们提出了一个方法来隔离和文化的小脑颗粒神经元祖细胞和产后小鼠小脑颗粒神经元。

产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

isolation-culture-post-natal-mouse-cerebellar-granule-neuron

Research

JoVE Journal

Biology

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

28.4K 次观看.  Columbia University. 在这里，我们提出了一个方法来隔离和文化的小脑颗粒神经元祖细胞和产后小鼠小脑颗粒神经元。

视频: Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

科研

生物学

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

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 ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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 ...