我们感谢亚历山大 Mourot 博士对手稿的评论。这项工作得到了 la 研究所 (2011 MALZ 003 01) 赠款的支持;ANR-15-CE16-0010 和 ANR-17-CE37-0010-03), 大人物是支持的奖学金从研究所阿尔茨海默氏症。我们感谢 IBPS 的动物设施 (巴黎, 法国)。

所有使用动物的实验程序都严格按照法国的规定进行 (农村 R214/87 R214/130), 符合欧洲经济共同体 (86/609/欧洲经济共同体) 和法国国家宪章的道德准则。动物实验伦理学研究。所有议定书均经查尔斯·达尔文道德委员会批准, 并提交法国教育部和研究部 (批准 2015 061011367540)。IBPS 动物设施由法国当局 (A75-05-24) 认证。
1. 初步考虑
注: 为避免污染, 在进行单细胞 rt-pcr 后, 应遵循以下建议。
<ol><li>不要在实验室中使用含有感兴趣基因的质粒, 因为它们是潜在的污染源。</li>
	<li>从凝胶电泳室预留一个实验台, 准备 PCRs。</li>
	<li>奉献一套吸管和气溶胶抗性过滤技巧, 以操纵 DNA 和 RNA 的浓度低于 1 ng/µL。不要用这些来分析 PCR 产品。</li>
	<li>始终使用 RNase 和 DNase 的产品, 戴上手套。</li>
	<li>使用专用化学品准备内部膜片钳解决方案。不要使用刮刀, 而是称量纸张来称量粉末。</li>
	<li>使用专用的 ph 电极和 ph 标准溶液, 因为它们可能是污染的源头 (例如, RNase)。</li>
	<li>贮存硼硅酸盐玻璃毛细血管;20µL 长, 细腻灵活的小贴士;20µL 微;自制的推出器 (贴在10毫升注射器上的补丁钳夹吸管);500µL PCR 管;10µL 气溶胶抗滤小贴士;和在专用框中的细长永久性标记 (图 1)。</li>
</ol>2. 底漆设计
注: 多路 rt-pcr 依赖两个放大步骤。在第一次 pcr 过程中, 所有感兴趣的基因都通过混合聚合 pcr 引物共同放大。为检测单细胞可靠的成绩单, 设计高效、选择性的 PCR 引物是必不可少的。使用嵌套 (内部) 引物的第二轮 PCR 提高了特异性和效率的放大。
<ol><li>检索 NCBI 参考序列数据库中感兴趣基因的被策划的 mRNA 序列32并且他们的相关的家庭 (https://www.ncbi.nlm.nih.gov/refseq/)。<ol>
<li>输入该基因的名称和感兴趣的种类作为查询, 并单击检索到的相关序列。</li>
		<li>为了限制对基因编码序列的分析, 点击发送到(右上角), 选择编码序列和FASTA 核苷酸作为格式。然后单击 "创建文件" 并使用 ". nt" 文件扩展名保存 FASTA 序列。</li>
	</ol>
</li>
	<li>使用金刚鹦鹉软件33 (多对齐构造 & 分析工作台) 或任何其他多对齐软件, 以确定同源区域。<ol>
<li>点击文件 |新建项目...并选择DNA作为序列类型。要导入基因序列, 请选择序列 |导入序列...并加载一个 ". nt" 文件。对所有相关序列重复该过程。</li>
		<li>单击并拖动 "示意图" 窗口中的所有序列以选择整个序列。</li>
		<li>通过选择对齐方式搜索同源区域 |搜索块...(ctrl+ S)。选择搜索方法中的线段对重叠。调整配对分数截止到一个相对较高的值 (例如, 1000) 和最小 seqs. 每块到要对齐的序列总数, 然后单击开始。</li>
		<li>在 "出现的搜索结果" 窗口中, 选择具有最高 MP 分数的结果, 然后单击 "链接"。如果找不到结果, 则减少每块的配对分数截止和/或最小 seqs.</li>
		<li>为促进同源区域的可视化, 选择对齐方式 |底纹 |平均得分。</li>
		<li>选择序列中未链接的部分, 然后重复步骤 2.2. 3–2.2 4 以潜在地确定其他与较低 MP 分数的同源区域。</li>
		<li>要获得最具体的引物, 请选择具有最少序列同源性的区域。</li>
	</ol>
</li>
	<li>获取所有拼接变体的序列并重复步骤 2.2. 1–2.2. 6。选择它们的共同区域以进行整体检测。考虑专用拼接变体分析20,34,35,36的替代卡带。</li>
	<li>使用37基因组 (基本局部对准搜索工具) 对动物模型基因组的 mRNA 序列进行排列, 以确定基因的内含子-外显子结构 (https://blast.ncbi.nlm.nih.gov/Blast.cgi)。<ol>
<li>通过单击鼠标并在 "输入查询序列" 部分中上载检索到的 FASTA 序列, 选择爆炸鼠标基因组。在程序选择中, 选择 "对高度相似的序列 (megablast)进行优化", 然后单击 "爆炸" 按钮。</li>
		<li>在 "说明" 部分中, 选择具有最高标识的对齐方式 (最多100% 个)。确保它对应于感兴趣的基因, 如在对齐部分的特征中所示。</li>
		<li>按查询起始位置在同一节中对齐方式进行排序, 以获得编码序列的外显子继承。记下所有命中的开始和结束位置, 这些点击大致对应于查询序列中内含子的位置 (图 2A)。</li>
	</ol>
</li>
	<li>选择两个不同的外显子的意义和反义引物, 以区分容易 cDNA 的基因组 DNA (gDNA) 放大的基础上的大小标准 (图 2)。 注: 当细胞核与细胞质一起收割时, gDNA 的扩增可能发生在低频。因此, 必须设计内含子 overspanning 底漆对 (图 2A)。在无内含子基因的情况下, 细胞核的收集是有问题的, 因为它可能导致潜在的混淆结果。为了解决这个问题, 包括一组引物, 旨在放大内含子序列, 以探测 gDNA25的存在。如果基因组控制是阳性的, 那么所有内含子的基因的结果必须被丢弃。</li>
	<li>为了实现有效的放大, 选择引物生成 amplicons, 其长度理想地由200和400基对组成 (图 2)。</li>
	<li>在多重 PCR 步骤中同时放大各种基因, 设计引物, 长度介于18和24核苷酸之间, 熔化温度 (Tm) 介于55和60摄氏度之间。</li>
	<li>为了尽量减少二次结构的形成, 降低放大率, 选择具有最小发夹和双工长度的感觉和抗感底漆 (图 2B)。</li>
	<li>使用相同的标准设计内部引物 (步骤 2.5–2.8)。</li>
	<li>验证 PCR 引物的特异性, 方法是将引物对准感兴趣生物体的参考 RNA 序列数据库, 使用核苷酸爆炸。<ol>
<li>在爆炸 (https://blast.ncbi.nlm.nih.gov/Blast.cgi), 点击核苷酸爆炸。在输入查询序列中粘贴底漆的序列。</li>
		<li>在 "选择搜索集" 部分中, 单击其他 (nr 等), 在数据库选项中选择参考 RNA 序列 (refseq_rna) , 并指定有机体 (例如, taxid:10090) ) 限制对所需物种的分析。</li>
		<li>在节目选择选择优选为有些相似的序列 (blastn)。在 "算法参数" 部分中, 将单词大小降低到 7, 以增加获取命中的机会。</li>
	</ol>
</li>
	<li>要设计选择性引物, 只保留那些序列至少有3不匹配的基因, 如果可能的话。</li>
	<li>在相对较高浓度的淡化底漆 (例如, 200 µM), 以确保所有外部引物可以混合在一起, 最终浓度为1µM。</li>
</ol>3. RT 试剂的制备
<ol><li>5x RT 混合: 在无 RNase 水中准备400µL 的工作 RT 混合溶液 (5x), 其中含有随机引物在25µM 和 dNTPs 2.5 毫米每。将此工作组合存储为20µL 整除数在500µL 管中。</li>
	<li>20x Dithiothreitol: 在 RNase 水中准备1毫升的0.2 米, 并将其存储为50µL 整除数。</li>
	<li>存储 2500 u 的 RNase 抑制剂 (40 u/µL) 和 1万 u 的逆转录酶 (RTase, 200 U/µL) 5 µL 整除数。</li>
	<li>为确保 RT 试剂的最佳质量, 请将整除数保存在-80 摄氏度。使用它们多达10个细胞, 只有一天。</li>
</ol>4. PCR 验证
<ol><li>通过对相对大量的总 RNA (通常为1µg) 进行反向转录, 制备基因, 从感兴趣的结构中提取38 。<ol>
<li>不要使用吸管专用于单细胞 rt-pcr 的这些初步步骤, 但使用无 RNase 吸管。稀释总 RNA 到1µg/µL。</li>
		<li>在500µL PCR 管, 添加1µL 稀释总 RNA 和8µL RNase 水。变性95摄氏度, 1 分钟, 冷却在冰管。</li>
		<li>添加4µL 的 rt 5x 缓冲器, 由制造商提供, 4 µL 的 rt 混合液, 1 µL RTase, µL 抑制剂 1 RNase, 1 毫米 (见 200)。</li>
		<li>轻拍管子, 旋转它, 在37摄氏度一夜之间孵化。</li>
		<li>储存的基因在-80 摄氏度, 长达数年。制备基因稀释到1整除/µL 的总 rna 当量。</li>
	</ol>
</li>
	<li>混合和稀释每一个底漆对在1µM 与吸管专用的单细胞 rt-pcr。使用20µL 的每个底漆组合为100µL PCRs。</li>
	<li>在冰上, 准备每个底漆对一个预混料包含: 水 (q.s., 80 µL), 10 µL 10X 缓冲器, 1 µL 100x dNTPs (每50µM), 500–1,000 pg 的 cDNA 稀释在 1 ng/µL, 0.5 µL (2.5 u, 5 u/µL) 的 Taq 聚合酶。</li>
	<li>使用 pcr 管紧密配合 pcr 机的水井, 以达到最佳温度控制。添加2滴 (~ 100 µL) 矿物油到预混料不接触壁或 PCR 管的上限。</li>
	<li>为了尽量减少底漆-脂肪酸的形成, 通过将 PCR 管放在 thermocycler 预加热95摄氏度, 进行热启动。三十年代后, 迅速排出20µL 的底漆混合在油顶部。</li>
	<li>在3分钟以后在95°c, 跑40个周期 (95 °c, 三十年代; 60 °c, 三十年代; 72 °c, 三十五年代) 后跟最后伸长步骤在72°c 为5分钟。</li>
	<li>用琼脂糖凝胶电泳 (2%, 重量/体积) 分析每种 PCR 产品10µL39。如果一些 PCR 产品没有预期的大小, 重新设计其他底漆 (见第2节)。 注意: 溴溴化铵是一种锂化学物质, 可以诱发 DNA 突变。使用前请咨询当地的安全办公室。操作时要戴手套。使用溴溴化 (10 毫克/毫升) 溶液的商业可用溶液, 而不是粉末, 以减少吸入的风险。同样, uv 光也会有害, 所以一定要戴上防紫外线防护眼镜或口罩。在专用于溴废料的容器中提取沾污的凝胶、小贴士、手套和缓冲器。</li>
	<li>一旦所有底漆对被单独验证, 测试多路协议 (图 3)。<ol>
<li>混合和稀释所有外部引物一起在1µM. 存储复合底漆混合在-20 摄氏度, 长达数周。</li>
		<li>在冰上, 准备一个预混料: 水 (q.s., 80 µL), 10 µL 10X 缓冲器, 1 µL 100x dNTPs (每50µM), 500–1,000 pg 的基因稀释在 1 ng/µL, 0.5 µL (2.5 u, 5 u/µL) 的 Taq 聚合酶。</li>
		<li>轻拍 PCR 管, 旋转, 并添加两滴 (100 µL) 矿物油。</li>
		<li>按照步骤4.5 中的说明进行热启动, 20 µL 复合底漆组合。在95摄氏度后3分钟, 运行 20 PCR 周期相同的步骤4.6。</li>
		<li>准备一些与分析的基因数量相同的 PCR 管。准备一个预混料, 在步骤 4.8.2, 但使用1µL 的第一 PCR 产品每基因作为模板。根据基因数量调整所有体积, 分析并考虑使用10% 的额外体积来补偿吹打错误。</li>
		<li>轻轻摇动, 旋转管, 在每个 PCR 管中分配80µL 预混料, 并添加两滴 (100 µL) 每管矿物油不接触壁或管帽。</li>
		<li>执行热启动 (请参见步骤 4.5), 将步骤4.2 中准备的内部底漆混合的20µL。运行 35 PCR 周期相同的步骤4.6。</li>
		<li>采用琼脂糖凝胶电泳技术对二次 PCR 产物进行分析。确保每一秒的 PCR 产生一个扩增子的预期大小。在低效或非特异性放大的情况下, 重新设计新的引物 (步骤 2.5–2.12) 并验证它们 (步骤 4.2–4.8)。</li>
	</ol>
</li>
</ol>5. 细胞内膜片钳溶液的制备和验证
注: 下面的协议描述了一种基于 K/葡萄糖酸盐的内部解决方案的制备和验证, 但几乎任何类型的补丁钳解决方案都可以使用, 只要它不妨碍 rt-pcr 的效率。佩戴手套是强制性的, 以获得一个无 RNase 的内部解决方案。
<ol><li>对于60毫升内贴钳记录解决方案, 准备即兴10毫升0.1 米的 KOH。</li>
	<li>溶解11.4 毫克 EGTA (0.5 毫米决赛) 在0.9 毫升 0.1 M KOH。</li>
	<li>添加40毫升 RNase 水, 溶解2.02 克葡萄糖酸盐 (144 毫米最终), 143 毫克的 HEPES (10 毫米决赛), 180 µL 1 米氯化镁2 (3 毫米决赛)。</li>
	<li>将 pH 值调整为 7.2, 加6毫升0.1 米的 KOH。</li>
	<li>调整渗透到 295 mOsm 13 毫升的 RNase 免费水。</li>
	<li>由于内部解决方案也被用作反向转录的缓冲器, 所以小心控制 Mg2 +浓度。在内部溶液中加入氯化镁2 , 以在 RT 反应中达到2毫米的最终浓度, 以补偿较低的镁2 +浓度。</li>
	<li>要在膜片钳录音后对收获的细胞进行标记, 请在上面描述的内部解决方案中添加2–5毫克/毫升 RNase biocytin (步骤 5.1–5.5)。</li>
	<li>过滤器 (0.22 µm 孔径) 内部解决方案, 并存储在-80 °c 100–250µL 整除数。</li>
	<li>为了验证单细胞 rt-pcr 的内部溶液的使用, 添加0.5 µL 的总 rna 稀释 1 ng/µL 到6µL 的补丁钳解决方案, 并留在板凳上约30分钟。</li>
	<li>与2µL 微, 添加2µL 的 RT 混合, 0.5 µL 的20x 滴滴涕, 0.5 µL RNase 抑制剂, 0.5 µL RTase, 并在37°c 隔夜孵化。</li>
	<li>旋转管子。冰上加水 (q.s., 80 µL), 10 µL 10X 缓冲器, 0.5 µL (2.5 u, 5 u/µL) 的 Taq 聚合酶。</li>
	<li>使用一组经过验证的底漆 (步骤 4.4–4.6) 执行40周期的 PCR。</li>
	<li>用琼脂糖凝胶电泳分析 PCR 产品 (见步骤 4.7), 验证其是否具有预期的大小。 注: 省略了总 RNA 的0.5 µL, 并用0.5 µL RNase 的水代替它, 将确保内部解决方案不含 rna 和/或 DNA 污染物。</li>
</ol>6. 急性切片制备
注: 本议定书描述了青少年 (即不到28天后) 雄性和雌性小鼠的切片程序。其他切割解决方案, 如蔗糖为基础的人工脑脊液 (aCSF) 也可用40。
<ol><li>准备2升的 aCSF 含 (毫米) 125 氯化钠, 2.5 氯化钾, 2 CaCl2, 1 氯化镁2, 1.25 NaH2PO4, 26 NaHCO3, 10 葡萄糖, 和15蔗糖。为减少谷氨酸在切片制备过程中的活性, 通过添加1毫米的 kynurenic 酸来制备切削液。</li>
	<li>切片前, 准备一个解剖套件, 包括手术剪刀, 细虹膜剪刀, 两个铲子, 镊子, 一盘纸过滤器, 和氰胶水。</li>
	<li>使用2/CO2 (95%/5%) 饱和的冰冷切削液, 并在-20 摄氏度冷却切割室。</li>
	<li>麻醉老鼠用一条小纸巾浸泡在异氟醚上。2分钟后, 确保鼠标在深度麻醉下, 通过验证没有对爪捏的反应。</li>
	<li>快速斩首鼠标。取出头皮, 打开头骨。仔细提取大脑, 把它放进一个小烧杯里, 里面装满了冰冷 (~ 4 °c), 用 O2/CO2进行了含氧的切割溶液。</li>
	<li>从-20 °c 的条件下取出切割室, 用纸巾除去湿气。</li>
	<li>仔细解剖大脑以隔离感兴趣的区域, 将其粘附在切割室中, 并加入 O2/CO2加氧的冰冷切割溶液。</li>
	<li>用 vibratome 切割300µm 厚片。在室温下将其转移到充满氧切割溶液的静止腔内, 并允许它们至少恢复0.5 小时。</li>
</ol>7. 单细胞 RT PCR 后贴片钳记录
<ol><li>定期清洗灌注系统与〜100毫升30% 小时2O2溶液, 并广泛冲洗与〜500毫升蒸馏水, 以避免细菌的增长, 因为它是被忽视的来源的 RNase 污染。</li>
	<li>氯化的纤维, 每收获一天, 用一个充满浓缩漂白剂的补丁吸管。不要使用微填充的补丁吸管, 而是一个20µL 长, 罚款, 灵活的提示附加到一个1毫升注射器。然后, 用 RNase 水冲洗, 用气体除尘器将其烘干。</li>
	<li>准备一小盒冰包含 5x RT 混合和20x 整除数。在台式冷却器中存储 RTase 和 RNase 抑制剂整除数-20 摄氏度。</li>
	<li>将切片转移到记录室, 并在 aCSF 中灌注。</li>
	<li>拉补丁吸管 (µm 开尖直径, 3–5 MΩ) 从硼硅酸盐玻璃戴手套, 并填补其中一个与 8 ul 内部解决方案。请记住, 吸管拉拔器的旋钮是 RNase 污染的源头。</li>
	<li>戴上新手套时, 把吸管放进吸管夹, 不要用手指接触灯丝。</li>
	<li>用正压接近贴片吸管, 并执行全细胞记录来表征目标细胞 (图 4)。</li>
	<li>为了保留基因, 将整个单元格配置中的时间限制为20分钟41.
</li>
	<li>在录音的末尾, 准备一个500µL PCR 管填充2µL 5x RT 混合和0.5 µL 的20x 数字电视, 旋转它, 并储存在冰上。</li>
	<li>应用温和的负压来收获细胞胞浆 (图 4)。视觉控制细胞的内容进入吸管, 同时保持密封紧密。<ol>
<li>如果考虑一些内含子较少的基因, 尽量避免收集细胞核。在这种情况下, 总要包括一组引物, 目的是放大 gDNA 以探测基因组污染。</li>
		<li>如果原子核靠近吸管尖端, 释放负压并将吸管移离细胞核。确保在新的吸管位置保存紧密封并重新启动以收集细胞质。</li>
	</ol>
</li>
	<li>停止收割时, 没有更多的材料来和/或在失去紧密封。</li>
	<li>轻轻地取出吸管, 形成外出斑块, 以限制细胞外碎片的污染 (图 4), 并有利于细胞膜的闭合, 随后进行组织化学分析。</li>
	<li>请记住, 旋钮, 并联, 电脑键盘, 或电脑鼠标是潜在的 RNase 污染的来源。如果他们在录音中被触摸, 更换手套。</li>
	<li>将吸管附着在推出器上, 并应用正压将其内容排出 PCR 管 (图 1)。</li>
	<li>将吸管的尖端分解成 PCR 管, 以帮助收集其内容。</li>
	<li>简要离心管, 添加0.5 µL 的 RNase 抑制剂和0.5 µL 的 RTase, 轻轻搅拌, 离心机, 并孵化过夜37摄氏度。</li>
	<li>旋转管和存储它在-80 °c 长达几个月, 直到 PCR 分析。</li>
	<li>通过添加水 (q.s.、80µL)、10µL 10x 缓冲器和0.5 µL (2.5 U) 的 Taq 聚合酶, 直接在含有10µL 的 RT 产品的管中执行第一放大步骤。然后, 按照步骤4.8 中描述的说明进行操作. 2–4.8. 8。</li>
</ol>8. 记录细胞的组织化学染色 (可选)
<ol><li>在电生理记录下, 维持20分钟的氧 aCSF, 使 biocytin 在轴突和树枝状树中扩散。</li>
	<li>将切片放在一个24井板井中, 并将其固定在4摄氏度, 在0.1 米磷酸缓冲液中用1毫升4% 多聚甲醛。</li>
	<li>清洗切片4次, 每5分钟, 1 毫升磷酸盐缓冲盐水 (PBS)。</li>
	<li>在同样的井中, permeabilize 和饱和的切片在1小时的室温与1毫升的 PBS 补充0.25% 海卫 X-100 和0.2% 明胶从冷水鱼皮肤 (PBS-GT)。</li>
	<li>一夜间孵化与荧光标记亲和稀释在 1/400 250–500µL PBS GT。</li>
	<li>清洗5次, 每5分钟, 与1毫升 PBS 和安装切片。</li>
</ol>

作者没有什么可透露的。

单细胞多重 rt-pcr 后贴片钳可以同时可靠地探测30多个基因在 electrophysiologically 识别细胞5的表达。在单细胞水平上分析基因表达需要高效的 PCR 引物。最限制的步骤之一是单元格内容的集合。其效率取决于贴片吸管尖端的直径, 它必须尽可能大, 同时匹配的细胞大小。吸管与1-2 µm 开尖直径被证明是适合大多数神经元类型。同样重要的是要确保只收集细胞的内容, 而不是周围的组织?...

大脑皮层包括多种细胞类型, 涉及各种生理过程。考虑到皮层细胞类型的形态学、生理和分子多样性, 它们的识别和表征是理解其特定功能的先决条件, 这是非常有挑战性的.1 ,2,3,4。
单细胞复合 rt-pcr 是基于膜片钳和 rt-pcr 技术相结合的。它可以同时探测30多个预定义基因在 electrophysiologically 识别细胞5中的表达。在记录吸管中加入神经元示踪剂进一步允许在组织化学揭示6、7、8、9后, 记录细胞的形态学特征,10. 基于对其表型性状5、9、10、11、12 的多元分析, 这是一种非常有用的神经类型分类方法. ,13,14。单细胞多重 rt-pcr 也适合于非神经细胞的特征, 如星形胶质细胞15,16,17, 几乎可以应用到每一个大脑结构18, 19、20、21、22、23和单元格类型, 假设它们可以在全单元格配置中进行记录。
这项技术是非常方便的识别细胞源和/或目标的传输系统7,8,15,16,20,21, 24,25,26,27,28, 特别是缺乏特定抗体时。它依赖于从视觉上识别出的细胞29的补丁钳录音, 因此也允许在特定的蜂窝环境8,15,16中定位细胞。此外, 由于大脑组织的细胞构筑被保存在脑切片中, 这种方法还可以研究具有神经元和非神经元元素的特征细胞的解剖关系7,8,18。
由于该技术受所收获细胞质数量和 RT 效率的限制, 在低拷贝数的情况下检测 mRNA 可能很难。虽然基于 RNaseq 技术的其他方法允许分析单个单元3、4、30、31的整个转录, 但它们需要高吞吐量的昂贵音序器不一定每个实验室都可以使用。由于单细胞复用 rt-pcr 技术采用终点 pcr 方法, 因此只需要广泛使用 thermocyclers。它可以很容易地开发在装有电生理设置的实验室, 不需要昂贵的设备。它可以在一天内提供一组预定义基因的定性分析。因此, 这种方法可以很容易地获得单个细胞的分子特征。

<table><tbody><tr><td>MACAW v.2.0.5</td><td>NCBI</td><td></td><td>Multiple alignement for primer design</td></tr><tr><td>Dithiothreitol</td><td>VWR</td><td>443852A</td><td>RT</td></tr><tr><td>Random primers</td><td>Sigma-Aldrich (Merck)</td><td>11034731001</td><td>RT</td></tr><tr><td>dNTPs</td><td>GE Healthcare Life Sciences</td><td>28-4065-52</td><td>RT and PCR</td></tr><tr><td>RNasin Ribonuclease Inhibitors</td><td>Promega</td><td>N2511</td><td>RT</td></tr><tr><td>SuperScript II Reverse Transcriptase</td><td>Invitrogen</td><td>18064014</td><td>RT</td></tr><tr><td>Taq DNA Polymerase</td><td>Qiagen</td><td>201205</td><td>PCR</td></tr><tr><td>Mineral Oil</td><td>Sigma-Aldrich (Merck)</td><td>M5904-5ML</td><td>PCR</td></tr><tr><td>PCR primers</td><td>Sigma-Aldrich (Merck)</td><td></td><td>PCR / desalted and diluted at 200 &amp;micro;M</td></tr><tr><td>Tubes, 0.5 mL, flat cap</td><td>ThermoFisher Scientific</td><td>AB0350</td><td>RT and PCR</td></tr><tr><td>BT10 Series - 10 &amp;micro;L Filter Tip</td><td>Neptune Scientific</td><td>BT10</td><td>RT and PCR</td></tr><tr><td>BT20 Series - 20 &amp;micro;L Filter Tip</td><td>Neptune Scientific</td><td>BT20</td><td>RT and PCR</td></tr><tr><td>BT200 Series - 200 &amp;micro;L Filter Tip</td><td>Neptune Scientific</td><td>BT200</td><td>RT and PCR</td></tr><tr><td>BT1000 Series - 1000 &amp;micro;L Filter Tip</td><td>Neptune Scientific</td><td>BT1000.96</td><td>RT and PCR</td></tr><tr><td>DNA Thermal Cylcer</td><td>Perkin Elmer Cetus</td><td></td><td>PCR</td></tr><tr><td>Ethidium Bromide</td><td>Sigma-Aldrich (Merck)</td><td>E1510-10ML</td><td>Agarose gel electrophoresis</td></tr><tr><td>Tris-Borate-EDTA buffer</td><td>Sigma-Aldrich (Merck)</td><td>T4415-1L</td><td>Agarose gel electrophoresis</td></tr><tr><td>UltraPure Agarose</td><td>Life Technologies</td><td>16500-500</td><td>Agarose gel electrophoresis</td></tr><tr><td>&amp;Phi;X174 DNA-Hae III Digest</td><td>NEB (New England BioLabs)</td><td>N3026S</td><td>Agarose gel electrophoresis</td></tr><tr><td>EDA 290</td><td>Kodak</td><td></td><td>Agarose gel electrophoresis</td></tr><tr><td>Electrophoresis Power supply EPS 3500</td><td>Pharmacia Biotech</td><td></td><td>Agarose gel electrophoresis</td></tr><tr><td>Midi Horizontal Elecrophoresis Unit Model SHU13</td><td>Sigma-Aldrich (Merck)</td><td></td><td>Agarose gel electrophoresis</td></tr><tr><td>Smooth paper with satin appearance</td><td>Fisherbrand</td><td>1748B</td><td>Patch clamp internal solution</td></tr><tr><td>Potassium Hydroxyde</td><td>Sigma-Aldrich (Merck)</td><td>60377</td><td>Patch clamp internal solution</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid</td><td>Sigma-Aldrich (Merck)</td><td>E3889</td><td>Patch clamp internal solution</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich (Merck)</td><td>H4034</td><td>Patch clamp internal solution</td></tr><tr><td>Potassium D-gluconate</td><td>Sigma-Aldrich (Merck)</td><td>G4500</td><td>Patch clamp internal solution</td></tr><tr><td>Magnesium chloride solution</td><td>Sigma-Aldrich (Merck)</td><td>M1028</td><td>Patch clamp internal solution</td></tr><tr><td>5500 Vapor Pressure Osmometer</td><td>Wescor</td><td></td><td>Patch clamp internal solution</td></tr><tr><td>Biocytin</td><td>Sigma-Aldrich (Merck)</td><td>B4261</td><td>Patch clamp internal solution</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich (Merck)</td><td>S5016</td><td>Slice preparation</td></tr><tr><td>D-(+)-Glucose monohydrate</td><td>Sigma-Aldrich (Merck)</td><td>49159</td><td>Slice preparation</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich (Merck)</td><td>S6191</td><td>Slice preparation</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich (Merck)</td><td>60128</td><td>Slice preparation</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich (Merck)</td><td>31437-M</td><td>Slice preparation</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich (Merck)</td><td>S5011</td><td>Slice preparation</td></tr><tr><td>Magnesium chloride solution</td><td>Sigma-Aldrich (Merck)</td><td>63069</td><td>Slice preparation</td></tr><tr><td>Calcium chloride solution</td><td>Sigma-Aldrich (Merck)</td><td>21115</td><td>Slice preparation</td></tr><tr><td>Kynurenic acid</td><td>Sigma-Aldrich (Merck)</td><td>K3375</td><td>Slice preparation</td></tr><tr><td>Isoflurane</td><td>Piramal Healthcare UK</td><td></td><td>Slice preparation</td></tr><tr><td>VT 1000S</td><td>Leica Biosystems</td><td>14047235613</td><td>Slice preparation</td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich (Merck)</td><td>H1009</td><td>Patch Clamp set-up cleaning</td></tr><tr><td>Thin Wall Glass Capillaries with filament</td><td>World Precision Instruments</td><td>TW150F-4</td><td>Patch Clamp</td></tr><tr><td>PP-83</td><td>Narishige</td><td></td><td>Patch Clamp</td></tr><tr><td>Eppendorf Microloader</td><td>Eppendorf</td><td>5242956003</td><td>Patch Clamp</td></tr><tr><td>BX51WI Upright microscope</td><td>Olympus</td><td></td><td>Patch Clamp</td></tr><tr><td>XC-ST70/CE CCD B/W VIDEO CAMERA</td><td>Sony</td><td></td><td>Patch Clamp</td></tr><tr><td>Axopatch 200B Amplifier</td><td>Molecular Devices</td><td></td><td>Patch Clamp</td></tr><tr><td>Digidata 1440</td><td>Molecular Devices</td><td></td><td>Patch Clamp</td></tr><tr><td>pCLAMP 10 software suite</td><td>Molecular Devices</td><td></td><td>Patch Clamp</td></tr><tr><td>10 mL syringe</td><td>Terumo</td><td>SS-10ES</td><td>Expelling</td></tr><tr><td>E Series with Straight Body (Holder)</td><td>Phymep</td><td>64-0997</td><td>Expelling</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich (Merck)</td><td>S7907</td><td>Histochemical revelation</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich (Merck)</td><td>S8282</td><td>Histochemical revelation</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich (Merck)</td><td>P6148</td><td>Histochemical revelation</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich (Merck)</td><td>X100</td><td>Histochemical revelation</td></tr><tr><td>Gelatin from cold water fish skin</td><td>Sigma-Aldrich (Merck)</td><td>G7041</td><td>Histochemical revelation</td></tr><tr><td>Streptavidin, Alexa Fluor 488 conjugate</td><td>ThermoFisher Scientific</td><td>S11223</td><td>Histochemical revelation</td></tr><tr><td>24-well plate</td><td>Greiner Bio-One</td><td>662160</td><td>Histochemical revelation</td></tr></tbody></table>

single cell multiplex reverse transcription polymerase chain reaction after patch-clamp

大脑皮层由多种细胞类型组成, 表现出多种形态、生理和分子特征。这种多样性阻碍了对这些细胞类型的识别和鉴定, 研究其特定功能的先决条件。本文介绍了多路单细胞反向转录聚合酶链反应 (rt-pcr) 协议, 它允许在切片的补丁钳记录后, 同时检测在单个细胞中的十个基因的表达。这种简单的方法可以进行形态学表征, 广泛适用于确定各种细胞类型的表型特征及其特定的细胞环境, 如血管附近。本协议的原则是用膜片钳技术记录一个细胞, 收获和反转转录其细胞质含量, 并通过多重 PCR 对预定义基因组的表达进行定性检测。它需要精心设计的 pcr 引物和细胞内贴钳解决方案兼容 rt-pcr。为了确保有选择和可靠的成绩单检测, 这项技术还需要适当的控制从细胞质采集到放大步骤。尽管这里讨论的预防措施必须严格遵循, 但几乎任何电生理学实验室都可以使用多重单细胞 rt-pcr 技术。

图 3显示了多路 rt-pcr 的代表性验证。该协议旨在同时探索12种不同基因的表达。水泡谷氨酸转运 vGluT1 被作为一个积极的控制谷氨酸神经元42。以 GABA 合成酶 (GAD65 和 67)、神经肽 Y (NPY) 和生长抑素 (SOM) 为 GABAergic 中间神经元3、5、11的标记物。cyclooxygenase-2 酶 (COX-2)...

Watch this Scientific Journal Video about Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp at JoVE.com

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

膜片钳后单细胞多重反转录聚合酶链反应

本协议描述了在膜片钳后执行单细胞多重反向转录聚合酶链反应所需的关键步骤和预防措施。该技术是一种简单有效的方法, 用于分析由膜片钳记录的单个细胞所确定的一组基因的表达谱。

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

single-cell-multiplex-reverse-transcription-polymerase-chain-reaction

Research

JoVE Journal

Neuroscience

9.7K 次观看.  Sorbonne Université. 本协议描述了在膜片钳后执行单细胞多重反向转录聚合酶链反应所需的关键步骤和预防措施。该技术是一种简单有效的方法, 用于分析由膜片钳记录的单个细胞所确定的一组基因的表达谱。

视频: Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

在使用单细胞RT - PCR技术的黑质神经元电压门控钾通道mRNA表达分析

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

科研

神经科学

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

计算机辅助多电极膜片钳系统

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

生物学

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

全细胞贴片记录用于电泳生理测定通道视紫质中的离子选择性

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

微电极阵列和膜片钳记录在人诱导多能干细胞来源心肌细胞上的技术应用

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

医学

Establishing a Whole-Cell Configuration through the Patch Clamp Method

Source: Segev, A. et al., <a href="https://app.jove.com/v/54024">Whole-cell Patch-clamp Recordings in Brain Slices.</a>&#160;J. Vis. Exp.&#160;(2016)
This video demonstrates the whole-cell patch clamp technique in a mouse brain slice. This technique establishes a stable connection between micropipette and neuron membrane, which is further disrupted to establish a whole-cell configuration to study the neuron&#39;s health and physiological responses.

通过膜片钳方法建立全细胞配置

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Slice ...