nih P01 HL112730 (抹布, JVE), nih R01 HL132075 (抹布, JVE), 芭芭拉史翠珊妇女心脏中心 (抹布, JVE), 桃乐茜和 e. 菲利普在分子心脏病学 (抹布) 的里昂椅子, 艾丽丝在妇女的心脏健康 (JVE) 和捷克科学院的椅子机构支持 RVO: 68081715 (MS)。

所有动物研究都是按照机构指南进行的, 并由雪松-西奈医学中心的机构动物护理和使用委员会批准。
1. Langendorff 灌注小鼠心脏
<ol><li>
小鼠心脏缺血再灌注 Langendorff 灌注的研究
	<ol>
<li>管理腹腔戊巴比妥钠70毫克/千克的成年小鼠 (8 周岁, 男性, C57BL6/j)。确认深部麻醉, 缺乏撤退到脚趾捏。</li>
		<li>Anticoagulate 腹腔肝素 500 U/千克。</li>
		<li>通过胸骨切口打开胸腔。打开隔膜, 沿肋边界切开至腋前轴。然后, 将前腋轴切开至前肢, 完全打开胸腔笼。之后, 视觉化的心脏, 钳位上升主动脉与钳和快速消费心脏。死亡是由于放血。将心脏放在冷的克雷布斯 (氯化钠6.9 克/升, 氯化钾0.35 克/升,NaHCO 3 2.1 克/升,MgSO 2 PO 4 0.16 克/升, , 4 克/升, 葡萄糖0.141 克/升和 CaCl2 2 克/升)。</li>
		<li>Cannulate 主动脉与钝尖针和灌注心脏 retrogradely 克雷布斯溶液在恒定压力模式。</li>
		<li>稳定期15分钟后, 心脏与全球无流性缺血30分钟, 其次再灌注30分钟。</li>
		<li>收获心后15分钟基线灌注后, 30 分钟缺血, 或30分钟再灌注后。修剪心房, 并在液氮中对组织进行冷冻。</li>
		<li>存储在-80 °c, 直到使用</li>
	</ol>
</li>
</ol>2. 组织均匀化、增溶和蔗糖密度梯度沉淀
<ol><li>
渐变准备
	<ol>
<li>准备2溶液 (15% 和 50%) 的100毫升蔗糖, 混合4毫升氯化钠 2.5 M; 0.8 毫升氯化镁2 1.25 M;1毫升三盐酸1米, pH 7.5;15克或50克蔗糖 (分别为15% 和 50%);超纯水达到100毫升;和二甲苯蓝 (0.02 毫克/毫升) 来着色15% 溶液</li>
		<li>过滤每个解决方案 (0.22 µm 过滤器)</li>
		<li>使用梯度的一天, 准备40毫升的每一个蔗糖溶液, 并添加放线菌酮到每个溶液, 以达到最终浓度的100µg/毫升<ol>
<li>使用渐变制作器来准备15% 和50% 蔗糖溶液的混合物</li>
			<li>用5毫升的15% 蔗糖梯度填充左侧隔间</li>
			<li>用50% 蔗糖梯度填充合适的隔间</li>
			<li>打开水龙头, 用磁力搅拌器将两个小隔间搅拌在泵上开关</li>
			<li>使用连接到第二个水龙头的管, 使混合蔗糖溶液流动在超离心管与薄壁。最终体积将约10毫升。</li>
		</ol>
</li>
		<li>保持梯度在4°c (一夜之间, 如果需要, 但不长)</li>
	</ol>
</li>
	<li>
心脏组织的均匀化
	<ol>
<li>融汇新鲜或冷冻心脏与 polytron 在过滤的溶解缓冲 (被改进为蔗糖梯度分馏) 包含100毫米氯化钾, 20 毫米三 pH 7.5, 5 毫米氯化镁2, 0.4% NP-40。包括放线菌酮100µg/毫升, 以维持 polysome 结构, 0.1 U RNase 抑制剂和蛋白酶抑制剂鸡尾酒 (1 片剂50毫升)。</li>
		<li>在冰上孵化15分钟的裂解液。</li>
		<li>离心机在 1.8万 x g 15 分钟在4°c 去除不溶性的材料并且收集上清。</li>
		<li>保留50µL 蛋白质测定、rna 分离和 rna 完整性分析 (见图 2)。</li>
		<li>将上清 (500–100µL) 层在梯度上, 平衡管与溶解缓冲。</li>
	</ol>
</li>
	<li>
沉淀和馏分的收集
	<ol>
<li>执行离心120分钟在 3.7万 rpm 在4°c 在摆动斗转子。根据制造商, 这对应于 22.8万 x g在最大半径。</li>
		<li>收集每一个梯度为17个分数 (大约600µL 每分数) 在连续监测吸收率在 254 nm (OD254)。</li>
		<li>按如下步骤对分数收集器进行编程:<ol>
<li>丢弃前3分钟的收集 (与在渐变前的管子中所含的液体相对应)</li>
			<li>从4到19分钟, 收集的分数以1毫升/分钟的速度在17个分数。</li>
		</ol>
</li>
		<li>把分数放在冰上, 一旦收集。如果没有立即处理, 请将样品冷冻-80 摄氏度。如图 1所示, 一个典型的 UV 密度对渐变的跟踪。</li>
	</ol>
</li>
</ol>3. Polysome 和 Nontranslating 馏分中基因的分离与分析
<ol><li>
mRNA 的提取
	<ol>
<li>解冻在冰上的分数, 如果他们收集后闪光冷冻</li>
		<li>将每个分数的200µL 转移到一个新鲜的管子上 注意: 在这个步骤中, 两个或多个连续的分数可以汇集在一起。在分析 OD254图的基础上, polysome 和非 polysome 分数不混合时, 应仔细进行。</li>
		<li>将 10 ng 荧光素酶 RNA 作为内部控制添加到每个样品中, 以达到规范化目的。 注: 荧光素酶底漆应作为内部控制, 以规范化的结果。</li>
		<li>添加700µL RNA 萃取试剂 (苯酚和异硫氰酸胍单相溶液; 见材料表) 到每管。混合好, 并孵化管5分钟的 RT (室温)。</li>
		<li>十五年代加入140µL 氯仿和涡流. 在 RT 中孵育2-3 分钟。</li>
		<li>离心样品15分钟在 1.2万 x g在4°c。</li>
		<li>小心地将上水相转移到新管上。</li>
		<li>由于分数的 RNA 含量不高, 增加10µg 糖原作为载体对每管。</li>
		<li>每管加350µL 异丙醇。拌匀, 在-20 摄氏度孵育1小时, 以提高产量。</li>
		<li>离心机为15分钟在 1.2万 x g在4°c。</li>
		<li>丢弃上清液, 加入700µL 乙醇75% 清洗 RNA 颗粒。</li>
		<li>离心机为5分钟在 7500 x g在4°c。</li>
		<li>去除乙醇, 让颗粒空气干燥10–15分钟。 注: 过度干燥的 RNA 颗粒将防止其在水中的完全溶解。</li>
		<li>并用重悬 RNA 颗粒在 50 ul RNase 无超纯 H2O。</li>
		<li>在55摄氏度孵化样品10分钟。</li>
		<li>使用每个样本的 2 ul, 以测量提取的 RNA 的质量和数量, 并存储其余的-80 °c。</li>
	</ol>
</li>
	<li>
反转转录和 qPCR
	<ol>
<li>使用 4 ul 的反向转录 cDNA 合成试剂盒 (见材料表) 的 20 ul 反应。根据样品的数量准备 supermix。将所需体积转移到每管, 并将输入 RNA 的 5 ul 添加到每个管中。这个体积可以被纠正, 以适用于 Taq 聚合酶工作条件的范围。</li>
		<li>在热循环仪中孵化管, 由制造商推荐的以下程序: 5 分钟在25°c, 20 分钟反向转录为20分钟在46°c, 并且逆转录酶失活为1分钟在95°c。</li>
		<li>运行 qPCR 与优化程序为每对引物。 注意: 要检测某些基因在分数中的存在, 应使用来自每个分数 (分离 RNA) 的相等体积来进行反向转录。</li>
	</ol>
</li>
	<li>
结果分析
	<ol>
<li>应用感兴趣基因和荧光素酶的 ct 值计算三角洲 ct 值</li>
		<li>将每个分数三角洲 Ct 值表示为所有分数中包含的总 mrna 值的百分比, 以可视化不同分数的 mrna 分布情况 (表 1)</li>
	</ol>
</li>
</ol>4. Polysome 馏分和 Nontranslating 馏分中 miRNAs 的分离与分析
<ol><li>
mRNA 和 miRNA 的提取 注意: 本协议遵循制造商的说明, 并进行了一些更改。<ol>
<li>解冻糖原和控制钉。保持冰块。</li>
		<li>添加700µL miRNA 萃取试剂到200µL 的汇集样品。</li>
		<li>添加1µg/µL 糖原, 并融汇它使用吸管。在室温下孵化5分钟。</li>
		<li>添加3.5 µL 1.6 x 108钉入控制和混合。</li>
		<li>添加200µL 氯仿和涡旋至少15秒。</li>
		<li>室温下孵育3分钟。</li>
		<li>离心机在 1.2万 x g 15 分钟在4摄氏度。</li>
		<li>使用吸管, 转移到上清到一个新的1.5 毫升管。丢弃中、下阶段。</li>
		<li>将1.5x 容积的100% 乙醇添加到上清液中并混合。</li>
		<li>将此混合物的700µL 转移到 miRNA 萃取塔上, 并在十五年代在 RT 上全速离心, 并丢弃流经;使用任何剩余的示例重复此步骤。</li>
		<li>将700µL 的缓冲1添加到该柱上, 并在十五年代在 RT 上全速离心;放弃流程。</li>
		<li>将500µL 的缓冲2添加到该柱上, 并在十五年代在 RT 上全速离心;放弃流程。</li>
		<li>在该柱上加入500µL 80% 乙醇, 并以全速将其离心为2分钟;放弃流程。</li>
		<li>将该列转移到新的2毫升名额管, 打开盖子, 并将其在 RT 上离心, 全速运行5分钟;放弃流程。</li>
		<li>添加16µL 的 RNAse 水到柱和离心机在全速1分钟的 RT. 保持洗脱液在冰上或存储在-80 °c, 以供将来分析。 注: 每个样品的两个微升可用于 OD 分析。</li>
	</ol>
</li>
	<li>
反向转录
	<ol>
<li>准备冰块。对于每20µL rt-pcr 反应, 添加4µL 的 miRNA rt 缓冲器, 2 µL 的核酸, 9 µL 的总 RNA, 2 µL 的酶和3µL 无 RNAse 水;混合它, 并简单地旋转下来。</li>
		<li>设置热循环仪如下: 37°c 为60分钟; 95°c 为5分钟; 4°c 举行。</li>
		<li>从热循环仪中取出管子, 加入200µL 的 RNAse 水。贮存在-20 摄氏度或进行实时 PCR。</li>
	</ol>
</li>
	<li>
实时 PCR 注: 本协议遵循96井板格式。在冰上准备反应混合物。<ol>
<li>根据协议所接受的范围, 准备 PCR 反应组合并添加 cDNA (从 miRNAs 以上)。多反应可以分批准备。</li>
		<li>在每个井中添加25µL 的 PCR 混合 + cDNA。</li>
		<li>用光学板密封密封板。</li>
		<li>在室温下用 1000 x g离心板1分钟。</li>
		<li>程序实时循环仪如下: 初始活化步骤在95°c 为15分钟; 3步循环: 变性在94°c 十五年代;退火在55°c 三十年代;延长在70°c 三十年代 (执行荧光数据收集);增加40个周期; 熔化曲线根据循环仪缺省。</li>
	</ol>
</li>
	<li>
比较分析
	<ol>
<li>规范化示例 (见表 2):<ol>
<li>查找 SCM 最小值。</li>
			<li>将所有 SCM 值规范化到最低限度。</li>
			<li>从 miRNAs 和参考基因 (REF) 的 Ct 值中减去此值。</li>
			<li>使用 2ΔCt公式分析数据。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. 蛋白质组分析
<ol><li>
polysome 中蛋白质的提取
	<ol>
<li>将所收集的蔗糖馏分组合成三个最终馏分。将分数标记为重(高效率 polysomes 在汇集的组分 4, 5, 6 和7以蔗糖的最高的集中; 梯度管的底部),光(低效率 polysomes 在汇集的分数 8, 9, 10 和 11; 中间的梯度管) 和非平移(汇集分数 12, 13, 14 和 15, 蔗糖浓度最低; 梯度管顶部)。对汇集的分数进行蛋白质检测。</li>
		<li>准备100% 股乙酸酸 (TCA), 并将其储存在4摄氏度在黑暗中。混合0.5 毫升的样品与60µL 100% TCA 和孵化在-20 °c 过夜在黑暗中。 注: 使用1.5 毫升低蛋白保留管 (见材料表)。</li>
		<li>夜间孵化后, 在冰上解冻样品, 离心机在 1.5万 x g, 4 °c 为30分钟。 注: 样品中总蛋白的100µg 在管底提供可见蛋白颗粒。</li>
		<li>预冷丙酮在-20 °c 冰箱。将0.5 毫升的冷丙酮添加到蛋白质颗粒和离心机 1.5万 x g, 4 °c 15 分钟。重复此步骤两次。</li>
		<li>空气干燥颗粒。并用重悬360µL 50 毫米三盐酸缓冲液中的颗粒 (pH 8)。 注: 如果需要, 使用脉冲 sonicator 并用重悬颗粒。</li>
		<li>添加40µL 0.1 米 dithiothreitol (最后浓度10毫米) 和孵化在55°c 在振动筛为45分钟. 添加50µL 0.15 米 iodoacetamide (最终浓度17毫米) 和在室温下孵化, 在黑暗中30分钟。</li>
		<li>准备胰蛋白酶溶液: 并用重悬20µg 的胰蛋白酶在100µL 50 毫米碳酸氢铵。在 1:50 (w/w) 胰蛋白酶中添加相应的胰蛋白酶溶液体积, 以确保 pH 值为 8, 并在37摄氏度的温度下在振动筛上孵育。 注: 添加1米碳酸氢铵, 使 pH 值为8。</li>
		<li>胰蛋白酶消化后, 冷却试样, 离心机在台式离心机上, 加入10% 甲酸对 pH 2–3, 以淬火胰蛋白酶活性, 并进行样品脱盐。 注: 使用µ洗脱板进行样品脱盐。</li>
		<li>在96井板井中, 用200µL 甲醇, 用真空三倍和200µL 0.1% 甲酸三次调理。用200µL 0.1% 甲酸三倍的样品进行试样清洗。洗脱样品与100µL 50% 乙腈/0.1% 甲酸两次到低蛋白保留0.5 毫升管。 注: 洗脱的样品首先使用引力, 其次是低真空。</li>
		<li>蒸发样品洗脱液干燥使用选矿厂, 或者直接进行质谱分析或存储在-80 °c, 直到使用。</li>
	</ol>
</li>
	<li>
质谱分析
	<ol>
<li>在50–100µL 中重建每个颗粒 (包括胰蛋白酶肽), 并向质谱仪注入1µL。 注: 优化重构和注入量以获得最大强度 LC/ms/毫秒信号。在我们的情况下, 最大信号 (总离子电流;TIC) 在扫描的心脏是在范围1E8 到2E9。</li>
		<li>使用陷印柱 C18 (300 µm id x 5 毫米, 5 µm, 100Å) 后, 使用 C18 柱 (75 µm 250 毫米, 2 µm, 100Å) 的肽分离, 流速设置为 300 nL/分钟. 将纳米源毛细管温度设置为275摄氏度, 喷雾电压为2伏。</li>
		<li>应用线性梯度 5–35% b 为90分钟, 35–95% b 为3分钟, 持有在 95% b 为7分钟和平衡在 5% b 为 25 min (a: 0.1% 甲酸/水和 B: 0.1% 甲酸在乙腈)。利用 CID 模式获取每 MS1 扫描的15种最高强度离子的 MS2 谱。使用3质谱复制为每个样品分析。 注: 优化和使用适用于特定质谱仪样品的实验条件和参数。第5.2 节所述的条件/参数。在实验室中得到了应用和优化。</li>
	</ol>
</li>
	<li>
蛋白质鉴定/定量
	<ol>
<li>在质谱分析后, 使用 MSConvert 软件 (http://proteowizard.sourceforge.net/tools/msconvert.html) 将原始光谱文件转换为兼容的 mzXML 文件。</li>
		<li>使用搜索条件将转换后的文件提交到 SEQUEST 搜索引擎,例如, UniProtKB/瑞士-波特鼠标数据库 (最新的规范化审查);半酶消化用胰蛋白酶 (在 KR/-之后);与2错过了分裂;前体质量范围: 400 到 4500;静态修改: carbamidomethyl (C) 57.021465;肽质量耐受性:50 ppm;片段质量类型: monoisotopic。 注: 可使用其他蛋白质数据库搜索引擎和数据管道。如果使用数据库 (如鼠), 它比鼠标和人的数据库小, 则可能需要搜索鼠标 (和/或人) 数据库以增加蛋白质组覆盖率。在这种情况下, 将需要删除蛋白质名称中的冗余。</li>
		<li>执行搜索后分析。设置用于查看数据的筛选器。例如, 将最小蛋白质概率 (蛋白质阈值) 设置为95% 或 99%,即,只有统计分析意味着在样本中存在95% 或99% 概率的蛋白质才会显示出来。设置用于肽阈值的筛选器 (例如, 95%),即在确定光谱是否识别肽时将使用的最小概率。选择将决定蛋白质是否存在的最小肽数量 (推荐2肽作为蛋白质鉴定的最低值)。</li>
		<li>评估确定的蛋白质质量/数量。确保 proteotypic 肽用于定量。如果异构体被确定, 确保它是根据观察的胰蛋白酶肽组成的氨基酸序列特有的特定异构体。任何 "类似的" 或假设的蛋白质应该进行比较, 看看观察到的肽是否对应于已知的蛋白质。</li>
	</ol>
</li>
</ol>

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</ol>

作者没有什么可透露的。

通过分析特定 mRNA 或整个转录6、7的平移状态, Polysome 剖面分析允许对蛋白质的翻译进行研究。当需要对本地翻译进行研究时, 如突触体8, 也有很大的帮助。传统上, 这种方法包括分离单和核糖体和相关的基因的蔗糖梯度, 可以结合基因组或蛋白质蛋白技术, 以获得预期结果6,9。例如, 我们小组?...

心脏以动态的方式对缺血 (I) 和再灌注 (R) 的损伤进行反应。然而, 在反应过程中, 对蛋白质合成的急性变化没有什么洞察力。为了解决这一问题, 我们利用 polysome 分析1的成熟方法来确定蛋白质丰度的变化, 反映核糖体和转化调节因子从细胞质到 polysomes 的再分配, 以及增加新合成的蛋白质 (NSPs)。在 I/R 的设置中, 新蛋白合成的增加发生在与新基因2的转录不一致的时间范围内;此外, 在 mRNA 表达水平和蛋白质丰度之间的不一致报告了3。基于这些原因, 我们选择分析蛋白质翻译所反映的动态蛋白质组的变化。为了做到这一点, 我们量化的 polysome 分数 mRNA, 并分析蛋白质组成的 polysome 分数。最后, 由于 microRNAs (大鹏湾) 调节基因的可用性, 并能干扰蛋白质翻译4、5的效率, 我们研究了大鹏湾在 polysome 馏分中的分布情况, 着重于对我/R 的响应。
我们选择使用孤立的小鼠 Langendorff 灌注模型和收获组织在基础条件下连续灌注, 30 分钟的全球无流性缺血后, 30 分钟的缺血后30分钟再灌注。然后, 我们可溶性的心脏组织和分离的 polysomes 在蔗糖梯度, 其次是蛋白质组分析和选择性检测基因和 miRNAs 的 PCR 和微阵列, 分别。这种方法的组合代表了一个强大的方法来了解动态蛋白质组, 使同时检测 mRNA, miRNA 和 NSPs, 以及调节蛋白, miRNA 和 mRNA 之间的 nontranslating 分数之间的重新分配,低效率的 polysomes 和高效的 polysomes (见图 1)。通过进一步分析诸如 eIF2α或 mTOR 等关键调控因素的磷酸化, 可以进一步深入研究这一过程的动态调节。现在详细描述了这些单独的步骤。

<table><tbody><tr><td>Pentobarbital</td><td>Vortech Phamaceuticals</td><td>9373</td><td>for euthansia</td></tr><tr><td>Heparin</td><td>Sagent</td><td>103424</td><td>used in langendorff preparation</td></tr><tr><td>forceps</td><td>Fine Science Tools</td><td>91110-10</td><td>used to hang the heart</td></tr><tr><td>Langendorff system</td><td>Radnoti + home made</td><td>n/a</td><td>A &#039;four heart&#039; system consisting of custom blown glass, tubing and water baths</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653-5KG</td><td>Krebs buffer and Sucrose gradient</td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td>Krebs buffer and Lysis buffer</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;42&lt;/sub&gt;</td><td>Sigma</td><td>P-5504</td><td>Krebs buffer</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M7774-500G</td><td>Krebs buffer</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G5767</td><td>Krebs buffer</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C1016-500G</td><td>Krebs buffer</td></tr><tr><td>Sucrose powder</td><td>Sigma</td><td>S0389-1KG</td><td>Sucrose gradient</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>208337</td><td>Sucrose gradient and Lysis buffer</td></tr><tr><td>Tris-base</td><td>Sigma</td><td>T1503-1KG</td><td>Sucrose gradient and Lysis buffer</td></tr><tr><td>Xylene Cyanole</td><td>Sigma</td><td>X-4126</td><td>Sucrose gradient</td></tr><tr><td>Cycloheximide</td><td>Sigma-aldrich</td><td>239763</td><td>Sucrose gradient and Lysis buffer</td></tr><tr><td>RNaseOUT</td><td>Life Technologies</td><td>C00019</td><td>RNAse inhibitor for Lysis buffer</td></tr><tr><td>Igepal CA-360 (NP40)</td><td>Sigma</td><td>I3021</td><td>Lysis buffer</td></tr><tr><td>Protease Inhibitor Cocktail tablets, EDTA free</td><td>Roche</td><td>5056489001</td><td></td></tr><tr><td>Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 mm x 89 mm</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>LE-80K</td><td>Ultracentrifugation of the gradients</td></tr><tr><td>Rotor</td><td>Beckman</td><td>SW41</td><td>Ultracentrifugation of the gradients</td></tr><tr><td>Biologic LP (pump)</td><td>Biorad</td><td>731-8300</td><td>Fractionation of the gradients</td></tr><tr><td>BioFrac</td><td>Biorad</td><td>741-0002</td><td>Fractionation of the gradients</td></tr><tr><td>Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube</td><td>Sigma</td><td>Z666513-100EA</td><td>Gradient fraction and RNA extraction</td></tr><tr><td>TRIzol Reagent</td><td>Life technologies</td><td>AM9738</td><td>RNA extraction</td></tr><tr><td>Luciferase Control RNA</td><td>Promega</td><td>L4561</td><td>RNA extraction</td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C606-4</td><td>RNA extraction</td></tr><tr><td>Glycogen, RNA grade</td><td>Thermo Fisher Scientific</td><td>R0551</td><td>RNA extraction</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>I9516</td><td>RNA extraction</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>E7023-1L</td><td>RNA extraction</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>BioRad</td><td>170-8891</td><td>Reverse transcription</td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>BioRad</td><td>175-5122</td><td>Quantative PCR</td></tr><tr><td>miRNeasy Micro Kit (50)</td><td>Qiagen</td><td>217084</td><td>Kit for total RNA isolation</td></tr><tr><td>miScript II RT Kit (50)</td><td>Qiagen</td><td>218161</td><td>Kit for miRNA reverse transcription</td></tr><tr><td>miScript Sybr Green PCR Kit (200)</td><td>Qiagen</td><td>218073</td><td>Kit for real-time PCR expression analysis of miRNAs</td></tr><tr><td>Centrifuge 5424R</td><td>Eppendorf</td><td></td><td>For centrifugation of 1.5 mL or 2.0 mL tubes at different temperatures. Max speed - 21130 x g</td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td></td><td>For real-time PCR plate centrifugation at different temperatures. Max speed - 2039 x g</td></tr><tr><td>My Cycler Thermal Cycler</td><td>Bio-Rad</td><td></td><td>For reverse transcription</td></tr><tr><td>CFX96 Real-Time System/C1000 Touch Thermal Cycler</td><td>Bio-Rad</td><td></td><td>For real-time PCR analysis</td></tr><tr><td>miRNeasy Serum/Plasma Spike-in Control</td><td>Qiagen</td><td>219610</td><td>For quality control of RNA isolation</td></tr><tr><td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear</td><td>Bio-Rad</td><td>HSP9641</td><td>For real-time PCR analysis</td></tr><tr><td>Microseal &#039;B&#039; PCR Plate Sealing Film, adhesive, optical</td><td>Bio-Rad</td><td>MSB1001</td><td>For real-time PCR plate sealing</td></tr><tr><td>Research plus Single-Channel Pipette, Gray; 0.5-10 &amp;micro;L</td><td>Eppendorf</td><td>UX-24505-02</td><td>For pipetting</td></tr><tr><td>PIPETMAN Classic Pipets, P20</td><td>Gilson</td><td>F123600G</td><td>For pipetting</td></tr><tr><td>PIPETMAN Classic Pipets, P200</td><td>Gilson</td><td>F144565</td><td>For pipetting</td></tr><tr><td>Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 &amp;micro;L</td><td>Gilson</td><td>17011782</td><td>For pipetting</td></tr><tr><td>Glycogen, RNA grade</td><td>Thermo Fisher Scientific</td><td>R0551</td><td>Improves total RNA isolation efficiency</td></tr><tr><td>Posi-Click 1.7 mL Tubes, natural color</td><td>Denville</td><td>C2170</td><td>RNA isolation and storage; reagent mix</td></tr><tr><td>Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically</td><td>Denville</td><td>C18098-4 (1000910)</td><td>Reverse transcription reaction</td></tr><tr><td>Sharp 10 Precision barrier Tips</td><td>Denville</td><td>P1096-FR</td><td>For pipetting</td></tr><tr><td>Sharp 20 Precision barrier Tips</td><td>Denville</td><td>P1121</td><td>For pipetting</td></tr><tr><td>Sharp 200 Precision barrier Tips</td><td>Denville</td><td>P1122</td><td>For pipetting</td></tr><tr><td>Tips LTS 1 mL Filter</td><td>Rainin</td><td>RT-L1000F</td><td>For pipetting</td></tr><tr><td>miScript Primer Assay (200)</td><td>Qiagen</td><td>(it changes according to the miRNA)</td><td>For real-time PCR analysis</td></tr><tr><td>Gradient Master ver 5.3 Model 108</td><td>BioComp Instruments</td><td></td><td>For preparation of sucrose gradients</td></tr><tr><td>trichloroacetic acid</td><td>Sigma Aldrich</td><td>T6399</td><td></td></tr><tr><td>acetone</td><td>Sigma Aldrich</td><td>650501</td><td></td></tr><tr><td>Tris hydrochloride</td><td>Amresco</td><td>M108</td><td></td></tr><tr><td>dithiothreitol</td><td>Fisher Scientific</td><td>BP172</td><td></td></tr><tr><td>iodoacetamide</td><td>Gbiosciences</td><td>RC-150</td><td></td></tr><tr><td>sequencing grade modified trypsine, porcine</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>ammonium bicarbonate</td><td>BDH</td><td>BDH9206</td><td></td></tr><tr><td>formic acid, Optima LC/MS</td><td>Fisher Chemical</td><td>A117</td><td></td></tr><tr><td>methanol, Optima LC/MS</td><td>Fisher Chemical</td><td>A454</td><td></td></tr><tr><td>acetonitrile, Optima LC/MS</td><td>Fisher Chemical</td><td>A996</td><td></td></tr><tr><td>Protein LoBind tubes 0.5 mL</td><td>Eppendorf AG</td><td>22431064</td><td></td></tr><tr><td>Protein LoBind tubes 1.5 mL</td><td>Eppendorf AG</td><td>22431081</td><td></td></tr><tr><td>HLB &amp;micro;Elution plate 30 &amp;micro;m</td><td>Oasis</td><td>186001828BA</td><td></td></tr><tr><td>SpeedVac concentrator</td><td>Thermo Scientific</td><td>Savant SPD2010</td><td></td></tr><tr><td>sonicator</td><td>Qsonica</td><td>Oasis180</td><td></td></tr><tr><td>centrifuge</td><td>Thermo Scientific</td><td>Sorvall Legend micro 21R</td><td></td></tr><tr><td>LC trap column PepMap 100 C18</td><td>Thermo Scientific</td><td>160454</td><td></td></tr><tr><td>LC separation column PepMap RSLC C18</td><td>Thermo Scientific</td><td>164536</td><td></td></tr><tr><td>mass spectrometer</td><td>Thermo Scientific</td><td>Orbitrap Elite ion trap mass spectrometer</td><td></td></tr><tr><td>MSConvert software</td><td>ProteoWizard Toolkit</td><td></td><td></td></tr><tr><td>Sorcerer-SEQUEST software</td><td>Sage-N Research, Inc.</td><td></td><td></td></tr></tbody></table>

dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion

蛋白质翻译中动态变化的研究需要专门的方法。在这里, 我们研究了新合成的蛋白质对缺血和再灌注反应的变化, 采用离体灌注小鼠心脏结合 polysome 分析。为了进一步了解蛋白质转化过程中的动态变化, 我们以胞内核糖体 (核糖体或 polysomes) 为基因, 并恢复了线粒体 polysomes, 并对其 mRNA 和蛋白质分布进行了比较。高效率的分数 (许多核糖体附着于 mRNA), 效率低下 (附着的核糖体较少), 也包括线粒体 polysomes 和非翻译分数。miRNAs 还可以与正在翻译的基因关联, 从而降低了翻译的效率, 我们研究了跨分数的 miRNAs 分布。对基因、miRNAs 和蛋白质在基础灌注条件下的分布进行了考察, 在30分钟的全球无流缺血和30分钟后再灌注。在这里, 我们介绍了用于完成这一分析的方法, 特别是从蔗糖梯度提取蛋白质的方法, 因为这一点以前没有描述过, 并提供了一些具有代表性的结果。

mRNA 分析 mrna 的结果可以表达为一个特定的 mRNA 在每一个分数的分布 (图 3A);为量化, 结合 polyribosomal 翻译分数和比较非翻译分数 (图 3B), 提出了 mRNA 丰度在翻译到 nontranslating 分数的比率。额外的信息是通过检查高效率的 polysome 分数分别从低效率 polysome 分数 (并与 nontranslating 分数分开) 获得。这在分析 miRNAs 时尤为重要, ...

Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

在这里, 我们提出了一个协议, 执行 polysome 分析的孤立灌注小鼠心脏。我们描述的方法, 心脏灌注, polysome 分析, 并分析 polysome 分数的基因, miRNAs, polysome 蛋白质组。

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

dynamic-proteomic-mirna-analysis-polysomes-from-isolated-mouse-heart

Research

JoVE Journal

Biochemistry

8.6K 次观看.  Cedars-Sinai Medical Center. 在这里, 我们提出了一个协议, 执行 polysome 分析的孤立灌注小鼠心脏。我们描述的方法, 心脏灌注, polysome 分析, 并分析 polysome 分数的基因, miRNAs, polysome 蛋白质组。

视频: Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

在分离的小鼠使用红桃心功能和能量学的评估 31 P核磁共振

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

科研

医学

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

遗传学

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

在疾病的染色质蛋白质组的定量分析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

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 ...

生物学

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

使用多体分析在发育中的鼠标大脑中进行翻译分析

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

神经科学

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

制备用于心肌梗死后成年小鼠心脏单细胞RNA测序的非心肌细胞悬液

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

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. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.