我们感谢Manfredo Quadroni，蛋白质分析设施和洛桑大学的电子显微镜设施的帮助。我们还要感谢H.G. Sprenger，K. Maundrell和Jourdain实验室的成员对手稿的建议和反馈。这项工作得到了Pierre-Mercier pour la Science基金会和瑞士国家科学基金会（项目资助310030_200796）的支持。

必须始终佩戴手套，并在层流罩下进行细胞培养步骤。将细胞保持在含有5%CO2的37°C培养箱中。该协议中提出的研究已获得批准，并按照洛桑大学和瑞士的动物使用指南进行。
1. RAW264.7巨噬细胞系的培养
<ol><li>在Dulbecco的改良鹰培养基（DMEM）中培养小鼠巨噬细胞RAW264.7细胞，高葡萄糖和谷氨酰胺补充有5%热灭活胎牛血清（HI-FBS）和100 IU / mL青霉素和100μg/ mL链霉素（P / S）。 注意：要分离线粒体，单个汇合的15厘米板（约70 x 10 7 RAW264.7细胞）就足够了。</li>
	<li>将RAW264.7细胞维持在组织培养板中。初始接种密度为 1 x 105 个细胞/mL 可在 3 天内形成汇合板。在培养基中使用 25 mL 细胞悬液用于 15 cm 平板。RAW264.7细胞具有高细胞分裂率，需要比大多数细胞系更频繁地分裂。</li>
	<li>分离 RAW264.7 细胞<ol><li>吸出培养基并用磷酸盐缓冲盐水（PBS）洗涤细胞一次。</li>
		<li>对于15cm板，加入8mL温热的RAW解离缓冲液（270mM氯化钾，30mM柠檬酸钠二水合物在H2O中，无菌过滤），并将细胞在37°C孵育5分钟。</li>
		<li>向平板中加入等量体积的培养基（解离缓冲液的1：1稀释），并移液器以分离和均质化细胞。</li>
		<li>将细胞悬液转移到锥形管中，并在室温下以300× g 离心管3分钟。</li>
		<li>吸出上清液并将沉淀重悬于适当体积的培养基（如步骤1.1中所述）中进行细胞计数。 注意：可以使用其他方法来分离RAW264.7细胞，例如胰蛋白酶或细胞刮刀。然而，这些方法对细胞更苛刻，并可能导致它们在分离后的几天内极化成M1样巨噬细胞。</li>
	</ol></li>
</ol>2. 骨髓来源巨噬细胞（BMDM）的分离和培养
注意：此处描述的协议适用于单个鼠标，可以针对多个鼠标进行放大。BMDM分离和培养的详细方案已在别处描述13，14。
<ol><li>用高剂量的CO2牺牲8-12周大的C57BL / 6小鼠。 注意：可以使用雄性或雌性小鼠。</li>
	<li>用75%乙醇喷洒鼠标进行消毒。</li>
	<li>解剖并收集小鼠的臀部、股骨和胫骨15.</li>
	<li>要从股骨和胫骨收集骨髓，请切除两块骨头的膝关节端15。通过切除髋臼从臀部恢复骨髓。</li>
	<li>将骨头转移到 50 mL 锥形管中，将 4 mL BMDM 培养基放在冰上（含有高葡萄糖和谷氨酰胺的 DMEM，补充有 5% HI-FBS、P/S 和 10 mM HEPES）。 注意：重要的是将骨骼保存在培养基中，以避免在解剖过程中骨髓干燥。</li>
	<li>在 6 孔板的两个不同孔中加入 4 mL PBS 和 4 mL 温热的 BMDM 培养基。</li>
	<li>将骨头和培养基从 50 mL 锥形管转移到 6 孔板的空孔中。</li>
	<li>使用一对镊子，将骨头转移到PBS井中清洗它们。</li>
	<li>将骨头转移到含有温BMDM培养基的孔中。</li>
	<li>用镊子在两个 0.5 mL 管的底部开一个直径为 1-2 mm 的孔，然后将它们放入 1.5 mL 管中。 注意：对于此快速步骤，无需将介质添加到管中。</li>
	<li>在每个 0.5 mL 管中，放置股骨、胫骨和髋关节，使暴露骨骼的骨髓朝向管底部。</li>
	<li>在室温下以 13，000 x g 离心管 1 分钟，以通过 0.5 mL 管的孔收集骨髓和剩余培养基并进入 1.5 mL 管中。丢弃带有骨头的 0.5 mL 管。</li>
	<li>将骨髓沉淀重悬于BMDM培养基中，并转移到15 mL锥形管中。</li>
	<li>加入高达 10 mL 的 BMDM 培养基。</li>
	<li>将 40 μm 细胞过滤器放在 50 mL 锥形管上，并通过它过滤骨髓悬浮液。</li>
	<li>在室温下以300× g 离心过滤的悬浮液5分钟，以恢复完整的细胞并从细胞悬液中去除小碎片。</li>
	<li>制备 70 mL BMDM 培养基，补充有 50 ng/mL 巨噬细胞集落刺激因子 （M-CSF）。</li>
	<li>将沉淀重悬于 10 mL 补充 M-CSF 的 BMDM 培养基中。</li>
	<li>将 9 mL 补充 M-CSF 的 BMDM 培养基添加到七个 10 cm 培养皿中的每一个中。</li>
	<li>在七个 10 cm 培养皿中的每一个中平板 1 mL 细胞（约 1 x 107 个细胞）。</li>
	<li>通过在平板上小心地上下移液，使每个板中的细胞悬液均质化，并将板转移到培养箱中。</li>
	<li>3 天后，向每个平板中加入 5 mL 补充有 50 ng/mL M-CSF 的温热 BMDM 培养基。</li>
	<li>3天后（第6天，骨髓分离后），通过显微镜检查BMDM的粘附和分化。 注意：此时，可以直接进行线粒体分离（步骤3）。或者，可以重新铺板BMDM，这允许用细胞因子和小分子处理它们。</li>
	<li>分离 BMDM<ol><li>从每个板中吸出培养基，并加入 7 mL 补充有 5 mM 乙二胺四乙酸 （EDTA） 的冷 PBS。</li>
		<li>将细胞在4°C孵育7-8分钟。</li>
		<li>使用 10 mL 移液器小心地上下移液，分离 BMDM。</li>
		<li>将来自所有七个板的重悬BMDM汇集到单个50 mL锥形管中，并在室温下以300 x g 离心3分钟。</li>
		<li>吸出上清液并将细胞重悬于40 mL温热的BMDM培养基中进行细胞计数。 注意：每只鼠标获得 7-9 x 107 BMDM 。对于蛋白质组学，建议至少使用 6 x 107 BMDM 进行线粒体分离。</li>
		<li>如果需要，根据实验目标对BMDM进行铺板和处理。如果没有，请直接执行步骤 3.3。</li>
	</ol></li>
</ol>3.差速离心制备粗线粒体级分
注意：在4°C下执行所有离心步骤。 需要两台离心机，一台带有外摆式转子和适配器，用于相对离心力至少为 300 x g 的锥形管，另一台相对离心力至少为 21，000 x g，适用于 1.5 mL 管。使用贴壁细胞时，请使用细胞刮刀。
<ol><li>对于贴壁细胞，吸出培养基并每 15 cm 板添加 10 mL PBS。 注意：在PBS中刮细胞可以同时清洗它们。如果细胞已经处于悬浮状态，请直接进入步骤3.3。</li>
	<li>使用细胞刮刀分离细胞，并将其汇集在单个 50 mL 锥形管中。通过上下移液使细胞悬液均质化。 注意：可以使用细胞刮刀分离细胞，因为这更快，并且很快就会裂解。</li>
	<li>对于每个实验条件，将5%的细胞悬液体积转移到1.5mL管中，并在室温下以300× g 离心5分钟。 注意：使用悬浮细胞时，请确保在之前用PBS清洗一次细胞，以去除培养基（如FBS）中可能的污染物。</li>
	<li>弃去上清液并将沉淀放在冰上。 注意：这将代表蛋白质组学的“总细胞”部分。</li>
	<li>将步骤3.1或3.2中的其余样品在室温下以300 ×g 离心5分钟。</li>
	<li>在冰上并使用冰冷的缓冲液执行以下所有步骤。</li>
	<li>吸出上清液并将细胞沉淀重悬于 5 mL 冰冷线粒体缓冲液 （MB）（210 mM 甘露醇、70 mM 蔗糖、10 mM HEPES/NaOH [pH 7.4] 和 1 mM EDTA）中。</li>
	<li>通过在4°C下以300× g 离心5分钟来回收细胞。</li>
	<li>将细胞沉淀重悬于0.5 mL冷MB中，并将其转移到1.5 mL管中。 注意：此过程产生的细胞浓度约为 1.5 x 10 8 BMDM 细胞/mL 或 3 x 108 RAW264.7 细胞/mL。</li>
	<li>使用装有 25 G 针头的 1 mL 注射器，将细胞悬液均质化 30 次（图 1A）。</li>
	<li>向试管中加入 1 mL 冷 MB 并通过倒置混合。在4°C下以2，000× g 离心匀浆细胞悬液5分钟。</li>
	<li>将 1 mL 上清液转移到冰上的新鲜 1.5 mL 管中，而不会干扰细胞沉淀。重悬细胞沉淀并再次均质化，如步骤3.7所示。</li>
	<li>将来自前两个步骤的匀浆细胞沉淀和上清液汇集在单个1.5mL管中，并在4°C下以2，000× g 离心5分钟。 注意：此时，沉淀主要包含细胞核和未破碎的细胞，并被丢弃。上清液含有细胞碎片，细胞质和细胞器，包括线粒体（图1B）。</li>
	<li>将上清液分配到四个 1.5 mL 管中。 注意：在此阶段将上清液分配到多个管中可改善以下步骤中污染物的去除。</li>
	<li>加入 MB 以使四个试管中每个管中的最终体积为 1 mL。倒置混合并在4°C下以13，000× g 离心管10分钟。 注意：在此步骤之后，可以看到具有两层的颗粒。底部，坚硬的棕色颗粒含有线粒体，并保留用于进一步纯化（图1C）。上部，松散的白色颗粒包含其他细胞结构，可以丢弃。</li>
	<li>请仔细执行此步骤。用尽可能多的白色上沉淀除去上清液。通过轻轻移液，可以重悬白色颗粒，然后将其丢弃，使棕色线粒体颗粒完好无损。</li>
	<li>将装有线粒体沉淀的四个试管中的一个放在冰上。这代表了蛋白质组学的“粗线粒体”部分。</li>
	<li>将其他三个沉淀汇集在 1.5 mL 管中，最终体积为 1 mL MB。</li>
</ol>4. 基于超顺磁性抗体的线粒体纯化
注意：在4°C的冷藏室中执行以下所有步骤。
<ol><li>将步骤 3.18 中的 1 mL 粗线粒体制剂转移到 15 mL 锥形管中，并加入 7 mL 补充有 150 mM NaCl （MB + NaCl） 的 MB。 注意：NaCl的添加可改善抗体结合并减少污染物与磁珠和线粒体的非特异性结合。</li>
	<li>将 50 μL Tomm22 珠子加入 8 mL 粗线粒体悬浮液中（图 1D），并将管在 4 °C 下在旋转轮上低速孵育 15 分钟。 注意：Tomm22磁珠与小鼠中产生的Tomm22单克隆抗体共价结合，与超顺磁珠偶联。</li>
	<li>同时，在磁铁上放置一根柱子。</li>
	<li>用 8 mL MB + NaCl 平衡色谱柱并丢弃流出物。</li>
	<li>在4°C下用Tomm22珠孵育样品15分钟后，将样品转移到色谱柱中。丢弃流出物。 注意：线粒体将保持附着在色谱柱中的磁珠上（图1E）。</li>
	<li>用 8 mL MB + NaCl 洗涤色谱柱 3 次。</li>
	<li>从磁铁上取下色谱柱，并将其放入 15 mL 锥形管中。</li>
	<li>通过向色谱柱中加入1.5 mL MB + NaCl来洗脱线粒体，并立即应用柱塞将纯化的线粒体洗脱到管中。</li>
	<li>将洗脱的线粒体转移到1.5mL管中，并在4°C下以21，000× g 离心10分钟。 注意：将形成棕色颗粒。其中包含分离的线粒体和一些抗体偶联珠（图1F）。</li>
	<li>小心地从沉淀中除去上清液。该沉淀代表蛋白质组学的“纯线粒体”部分。该颗粒与步骤3.4和3.17中的颗粒一起可以储存在-20°C，并准备用于下游应用。</li>
</ol>

使用两步线粒体分离改进蛋白质定量

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MITO-Tag+mice+enable+rapid+isolation+and+multimodal+profiling+of+mitochondria+from+specific+cell+types+in+vivo.">MITO-Tag mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo.</a> Proceedings of the National Academy of Sciences. 116 (1), 303-312 (2019).</li><li>Nusinow, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+of+the+cancer+cell+line+encyclopedia.">Quantitative proteomics of the cancer cell line encyclopedia.</a> Cell. 180 (2), 387-402 (2020).</li><li>Iuso, A., Repp, B., Biagosch, C., Terrile, C., Prokisch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+bioenergetics+in+isolated+mitochondria+from+various+mouse+tissues+using+seahorse+XF96+analyzer.">Assessing mitochondrial bioenergetics in isolated mitochondria from various mouse tissues using seahorse XF96 analyzer.</a> Methods in Molecular Biology. 1567, 217-230 (2017).</li><li>Hornig-Do, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+functional+pure+mitochondria+by+superparamagnetic+microbeads.">Isolation of functional pure mitochondria by superparamagnetic microbeads.</a> Analytical Biochemistry. 389 (1), 1-5 (2009).</li><li>Liao, P. C., Bergamini, C., Fato, R., Pon, L. A., Pallotti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria+from+cells+and+tissues.">Isolation of mitochondria from cells and tissues.</a> Methods in Cell Biology. 155, 3-31 (2020).</li><li>Lanza, I. R., Nair, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+isolated+mitochondria+in+vitro.">Functional assessment of isolated mitochondria in vitro.</a> Methods in Enzymology. 457, 349-372 (2009).</li></ol>

作者没有什么可透露的。

我们结合了差速离心和免疫捕获，以提高线粒体分离的纯度。我们的程序允许访问用于鉴定和表征新型线粒体蛋白的主要材料。该方案简单而稳健，可以应用于细胞系、原代细胞和组织，而无需进行基因改造。我们通过对整个纯化过程中不同阶段采集的样品进行免疫印迹和蛋白质组学分析来验证我们的方案。
与单一分离方法相比，不同性质的富集步骤（此处为离心和免疫标记）的组合可生成更强大的方案来分离线粒体。这是因为，虽然线粒体蛋白在两次纯化中都会富集，但在两次富集步骤后，污染物也不太可能富集。虽然密度梯度超速离心也可以实现高线粒体纯度，但这种方法需要大量的起始材料和超速离心机。最后，与最近基于基于标签的线粒体分离20的方法相比，我们的方法不需要对样品进行基因修饰，使其适用于任何来源的主要材料。
在应用我们的协议时，在实验设计中需要考虑一些技术和生物学因素。（1）起始材料的量对于获得足够的材料至关重要。不可避免地，在匀浆过程中（步骤3.10）会丢失少量线粒体，因为并非所有细胞都被裂解，或者在三柱洗涤期间（步骤4.6）。虽然我们的方案侧重于纯度而不是产量，但线粒体分离的效率及其产量尚未得到测量或优化。使用更多的Tomm22珠和更多的色谱柱有望提高线粒体回收率。同时，对均质化步骤进行彻底优化也可以提高线粒体产量。该协议以及我们在此处报告的RAW264.7细胞和BMDM的初始细胞数足以用于蛋白质组学，并且可以针对其他应用进行调整。在原代BMDM的情况下，我们发现一只小鼠足以进行一次复制。必要时，可以扩大该程序以从多种动物中分离BMDM，然后可以将其汇集以获得足够的材料。可以根据细胞类型、大小和线粒体含量优化细胞数量。（2）Tomm22在所有细胞类型和组织的线粒体上表达21，但其表达水平可能会有所不同。因此，在设计比较不同条件的实验时，重要的是要确保Tomm22的表达水平具有可比性。此外，由于Tomm22的普遍表达，不可能研究复杂组织中的细胞类型特异性线粒体蛋白。（3）产生纯线粒体所需的时间（约2.5小时）与瞬时事件的研究不相容，例如代谢谱的变化。在这种情况下，我们建议直接基于标签的免疫捕获9，这也允许研究细胞类型的特异性线粒体 in vivo20.（4）尽管单独使用Tomm22抗体标记的磁珠获得的分离线粒体的研究在功能测定中显示出活性11，还有待确定使用我们的协议生成的线粒体是否与下游基于活性的测定兼容。MitoTracker或四甲基罗丹明甲酯高氯酸盐（TMRM）染色或呼吸测量是量化分离线粒体功能的潜在方法22.（5）从色谱柱中洗脱“mito-pure”样品后，一些Tomm22珠将存在于纯线粒体部分（图2B).虽然我们观察到胰蛋白酶消化和蛋白质质谱没有干扰，但在其他下游应用中应考虑这些磁珠和免疫球蛋白的存在。Tomm22抗体是在小鼠体内产生的单克隆抗体23，因此重要的是要记住，当在免疫印迹中使用针对小鼠的二抗时，它将在免疫球蛋白链的大小上产生非特异性条带。（6）细胞悬液的完全均质化是成功分离线粒体的关键。在这里，我们使用带有25 G针头的注射器来裂解RAW264.7细胞和BMDM。然而，根据细胞类型及其大小，其他机械均质方法（例如使用Dounce均质器）或更受控的方法（例如细胞均质器设备）可能更合适。也可以考虑非机械均质方法，例如温和超声处理。组织均质化方法在其他研究中进一步讨论24,25.（7）尽管免疫印迹验证是最直接和更便宜的方法，但其结果可能并不总是与整个细胞器水平的变化相关。这就是为什么我们建议使用蛋白质组学来完全验证线粒体和其他细胞器的富集或消耗。
这里描述的两步线粒体纯化方案使我们能够生成线粒体纯度增加的顺序样品，这使我们能够通过减法蛋白质组学发现新的线粒体蛋白候选物12。对于我们的分析，我们使用严格的阈值来选择显着富集的线粒体蛋白，尽管这可能无法识别一些已知的线粒体蛋白（图3A），但新线粒体蛋白发现的假阳性率降低。然而，重要的是要强调，我们的协议揭示的任何候选蛋白质都必须通过正交方法进行验证。我们建议使用羧基末端GFP标记或使用针对内源性蛋白的抗体，以通过显微镜或蛋白酶保护测定验证与线粒体的关联。
在未修饰的细胞和组织的情况下直接应用我们的方法提供了一个强大的工具来研究线粒体在健康和疾病条件下如何改变和适应其环境。将我们的方案应用于细胞系，动物疾病模型，人体体液，甚至手术活检可能被证明对增强我们对线粒体及其相关疾病的理解特别有用。

线粒体是复杂而动态的细胞器，能够感知和适应细胞的代谢需求。线粒体是细胞代谢复杂性的核心，是碳水化合物、蛋白质、脂质、核酸和辅因子代谢反应汇聚的代谢枢纽1。它们还充当先天免疫反应途径的信号细胞器，并响应离子和活性氧的变化2，3。迄今为止，大约有1，100种蛋白质被映射到线粒体4，5，6，但我们可以假设还有更多的蛋白质有待发现，特别是那些仅在某些细胞类型中表达或在特定环境条件下短暂表达的蛋白质。开发量化感兴趣的代谢状态下线粒体组成变化的新方法将增加我们对这些细胞器的了解，并突出以线粒体功能障碍为特征的疾病的新治疗途径7。
目前，有不同的线粒体分离方案可用，具有不同的产量和纯度水平8。基于离心的方法因其简单性和低成本而最受欢迎。虽然适用于大多数应用，但当使用更复杂的基于密度梯度的应用时，差速离心的缺点是线粒体纯度较低，并且需要大量起始材料。近年来，出现了分离线粒体的新方法，例如基于标签的免疫捕获（“MITO-IP”）9 和荧光激活细胞器分选10。尽管这两种程序都可以生成高纯度的样品，但前者需要基因工程来标记线粒体以进行亲和纯化，这使得该方案与来自未改性生物体或人类供体的主要材料不兼容。同时，后者依赖于流式细胞术和分选仪器的可及性。结合不同的分离方法，有望产生更稳健的实验方案和更高的纯度。
在这里，我们提出了一种基于两种现有方法组合的线粒体分离新方案：（1）差异离心以分离粗线粒体部分，以及（2）线粒体的无标签免疫捕获，超顺磁珠与外线粒体膜22（Tomm22）11（一种无处不在的线粒体外膜蛋白）的转位酶抗体共价结合（图1).我们描述的程序与定量蛋白质质谱兼容，并且由于它是无标签的并且不需要遗传操作，因此可以应用于广泛的研究模型，从细胞系到体液再到整个动物组织。此外，在协议中使用两个步骤可以使用减法蛋白质组学6，12 来发现新的线粒体蛋白并研究其表达。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD Plastipal</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G Needle</td>
			<td>BD Microlance</td>
			<td>300400</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TOM22 Microbeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-127-693</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>FisherScientific</td>
			<td>11577692</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX</td>
			<td>ThermoFisher</td>
			<td>31966</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>FisherScientific</td>
			<td>BP-120-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>BioConcept</td>
			<td>5-31F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns and plungers</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage colony-stimulating factor</td>
			<td>Immunotools</td>
			<td>12343115&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>71380</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>BioConcept</td>
			<td>4-01F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Corning</td>
			<td>BH93B-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline 10X</td>
			<td>Eurobio Scientific</td>
			<td>CS3PBS01-01</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Vi-CELL BLU Cell Viability Analyzer</td>
			<td>Beckman Coulter</td>
			<td>C19196</td>
			<td></td>
		</tr>
	</tbody>
</table>

two-step tag-free isolation of mitochondria for improved protein discovery and quantification

大多数生理和疾病过程，从中枢代谢到对神经变性的免疫反应，都涉及线粒体。线粒体蛋白质组由1000多种蛋白质组成，每种蛋白质的丰度可以响应外部刺激或疾病进展而动态变化。在这里，我们描述了一种从原代细胞和组织中分离高质量线粒体的方案。两步程序包括（1）机械均质和差速离心以分离粗线粒体，以及（2）线粒体的无标签免疫捕获以分离纯细胞器并消除污染物。通过定量质谱分析每个纯化阶段的线粒体蛋白，并计算富集产量，从而通过减法蛋白质组学发现新的线粒体蛋白。我们的实验方案为研究细胞系、原代细胞和组织中的线粒体含量提供了一种灵敏而全面的方法。

在本协议中生成三种线粒体纯度递增的样品：总细胞，粗线粒体（“mito-purde”）和纯线粒体（“mito-pure”）（图1）。我们通过在凝胶上加载每个级分的等量蛋白质并进行免疫印迹验证了RAW264.7巨噬细胞系线粒体的纯化，发现线粒体柠檬酸合酶（Cs）在每个纯化步骤中富集;同时，来自细胞质（GAPDH），质膜（Na / K ATPase），细胞核（Lamin B），溶酶体（Lamp1）和内质网（ER）（Pdi）的蛋白质逐渐消失（图2A）。使用BMDM获得了类似的结果。为了进一步验证分离的线粒体的纯度和完整性，对纯线粒体部分进行了电子显微镜检查。我们观察到线粒体具有经典的椭圆形和完整的嵴，周围环绕着对应于抗体包被珠子的电子致密颗粒（图2B）。因此，可以得出结论，我们的协议丰富了线粒体，耗尽了其他细胞成分，并保持了线粒体结构的完整性。
接下来，使用液相色谱与质谱联用（LC / MS）对每个馏分进行蛋白质组分析。从总细胞中提取的提取物中总共鉴定出6，248种蛋白质，其中907种先前在MitoCarta3.0清单5中被注释为线粒体。在过滤阈值至少为两种独特肽的蛋白质后，我们根据每个样品中每种蛋白质的强度与总细胞的比较计算了每种蛋白质的富集评分。然后，我们使用基因本体（GO）16，17和MitoCarta3.05作为参考，将蛋白质分配到七个主要的亚细胞区室：线粒体，ER，溶酶体，高尔基体，细胞骨架，细胞核和细胞质。重要的是，观察到线粒体蛋白在粗线粒体和纯线粒体部分中的平均富集分别超过10倍和20倍以上（图2C）。相比之下，分析的其他六个细胞区室的组分在纯化过程中耗尽。特别值得注意的是，在粗线粒体部分中，我们观察到ER和溶酶体蛋白的瞬时富集，这两类污染物蛋白在差异离心方案18之后经常出现。这可能是由于细胞器-细胞器相互作用和相似的沉降系数，特别是溶酶体，其在巨噬细胞中含量很高19。虽然两者都在免疫捕获后大部分耗尽，但我们检测到来自丝分裂纯部分ER-线粒体接触位点的蛋白质的小信号。
然后，我们直接比较了来自总细胞和丝分裂纯样品的蛋白质丰度，并观察到两个不同的群体，对应于线粒体和非线粒体蛋白质（图2D）。虽然绝大多数MitoCarta蛋白聚集在一起，但我们发现一些（<5%）与非MitoCarta蛋白聚集在一起。这些蛋白质可能代表（1）胞质线粒体相互作用蛋白（MitoCarta3.0版本中注释的新类别），（2）双定位蛋白，或（3）错误注释的蛋白质。相反，我们发现了一些非MitoCarta蛋白与线粒体蛋白聚集的实例。虽然这些蛋白质可能代表分离过程的污染物，但它们也可能代表以前未被归类为存在于线粒体中的蛋白质。
为了研究这一类新的潜在线粒体蛋白，使用了减法蛋白质组学，这种方法已被证明对发现细胞器蛋白质组（包括线粒体6，12）有用。减法蛋白质组学假设线粒体在纯化步骤中应该变得富集，污染物应该被耗尽6。例如，虽然污染物可能在差异离心（例如，由于相似的沉降特性）或免疫捕获期间（例如，由于非特异性抗体结合）期间积累，但只有 真正的 线粒体蛋白应该在两者中显着积累。因此，可以过滤掉在纯线粒体部分中发现但显示出不一致的富集模式的蛋白质。在RAW264.7细胞的本例中，通过为丝裂粗和丝分裂纯样品设置≥1的独特肽阈值，并使用严格的富集阈值，我们能够从差异离心后最初在粗线粒体级分中发现的1，127种蛋白质中细化回收的线粒体蛋白质组列表，在使用Tomm22免疫选择进行第二轮纯化后，细化到481种蛋白质。丝图纯级分中MitoCarta注释蛋白数量的减少反映了对选择的高度严格性。有趣的是，Mito-pur部分中存在的70种蛋白质在MitoCarta3.0清单中不存在（图3A，B）。后一种蛋白质可能代表潜在的新型线粒体候选蛋白质，其可能仅在RAW264.7巨噬细胞系和相关细胞中表达，值得进一步研究。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65252/65252fig01v2.jpg"> 图 1：两步无标签线粒体分离方案的图示。 （A）通过25G针头破坏细胞悬液。（B）通过以2，000×g离心分离细胞核和整个细胞，并保存上清液。（C）粗线粒体通过上清液在13，000×g（mito-粗）下的差异离心分离。（D）然后将粗线粒体与与超顺磁珠共价连接的Tomm22抗体（Ab）一起孵育。（E）使用磁柱将线粒体-Tomm22抗体-珠复合物与污染物分离并洗脱。（F）通过离心（水线纯）收集并浓缩纯线粒体。<a href="https://www.jove.com/files/ftp_upload/65252/65252fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65252/65252fig02v2.jpg"> 图2.从两个巨噬细胞来源分离线粒体的代表性结果。 （A） 使用线粒体柠檬酸合酶（Cs - 线粒体）、甘油醛 3-磷酸脱氢酶（Gapdh - 细胞质）、钠钾泵（Na/K ATP 酶 - 质膜）、层粘连蛋白 B（层粘连蛋白 B - 细胞核）、溶酶体相关膜蛋白 1（Lamp1 - 溶酶体）和蛋白质二硫醚异构酶 （Pdi - ER） 的抗体对 RAW264.7（顶部）和 BMDM 细胞（底部）进行蛋白质免疫印迹分析。（B）从RAW264.7细胞纯化的线粒体的电子显微镜检查。线粒体周围的高密度颗粒对应于从色谱柱中洗脱后与丝图纯样品一起进行的Tomm22珠。比例尺：80 nm。（C） RAW264.7 细胞中七个细胞区室的总细胞、丝氨酸粗和丝氨酸纯的富集评分。MitoCarta3.0和GO用于蛋白质注释，并表示平均分数。缩写：ER = 内质网。（D）来自RAW264.7细胞的总细胞和丝线纯样品中蛋白质的蛋白质丰度值（riBAQ）。MitoCarta3.0蛋白以橙色显示。 <a href="https://www.jove.com/files/ftp_upload/65252/65252fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65252/65252fig03v2.jpg"> 图3.使用减法蛋白质组学发现新型线粒体蛋白。 （A）用于发现新型线粒体蛋白的减法蛋白质组学策略。应用高选择阈值（4x 和 2x）以最大程度地减少误报的选择。（B）先前未在MitoCarta3.0清单中注释的新线粒体候选蛋白的富集产量（总细胞的倍数）。 <a href="https://www.jove.com/files/ftp_upload/65252/65252fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification at JoVE.com

作者聚焦：两步法无标签线粒体分离，改进蛋白质发现和定量  

我们提出了一个用于高质量线粒体分离的两步方案，该方案与蛋白质组规模的蛋白质发现和定量兼容。我们的协议不需要基因工程，因此适用于研究来自任何原代细胞和组织的线粒体。

线粒体的两步无标签分离，用于改进蛋白质发现和定量

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial ...

我们的实验室研究线粒体和能量代谢的变化，以及这些变化如何影响健康和病理过程，如免疫激活、癌症或代谢紊乱。我们的目标是通过更好地了解线粒体功能和能量代谢，帮助开发针对各种疾病的新疗法。基于质谱的蛋白质组学、代谢组学和CRISPR-Cas9基因筛选等工具正变得越来越流行，用于识别特定细胞类型和环境中线粒体功能和能量代谢的分子机制。与其他方法相比，我们的方案采用两个纯化步骤，差示离心和免疫捕获进行线粒体分离。这提供了几个优点，例如改善污染物的去除，并且它也是识别新线粒体蛋白质的强大工具。我们的协议提供了对组成以及不同细胞类型和环境中线粒体功能的见解。这可以帮助我们更好地了解免疫激活等正常过程，以及癌症等病理过程。我们将专注于线粒体巨噬细胞。我们将测试线粒体的组成在免疫反应期间如何变化，以更好地了解它们的功能。我们的发现可能导致具有例如抗炎作用的药物的开发。

two-step-tag-free-isolation-mitochondria-for-improved-protein

Research

JoVE Journal

Biology

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

5.4K 次观看.  University of Lausanne. 我们的实验室研究线粒体和能量代谢的变化，以及这些变化如何影响健康和病理过程，如免疫激活、癌症或代谢紊乱。我们的目标是通过更好地了解线粒体功能和能量代谢，帮助开发针对各种疾病的新疗法。基于质谱的蛋白质组学、代谢组学和CRISPR-Cas9基因筛选等工具正变得越来越流行，用于识别特定细胞类型和环境中线粒体功能和能量代谢的分子机制。与其他方法相?...

视频: 作者聚焦：两步法无标签线粒体分离，改进蛋白质发现和定量  

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

科研

生物学

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

人肝细胞通过一个两步胶原酶灌注过程隔离

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换：应用程序的设计和开发非ATP竞争性CDK抑制剂

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

一种改进的小鼠原代肝细胞分离时间和劳动效率方案

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...