我们感谢麻省大学的流式细胞仪的核心是:理查德Konz，特德Giehl，芭芭拉Gosselin，粤华谷和塔米Krupoch。这项工作是由美国国立卫生研究院/ NHLBI RO1 HL084168（MS）和美国国立卫生研究院CA的T32 - 130807（JRS）。也可用于糖尿病内分泌研究中心授予DK32520支持的核心资源。

1。收获的组织<ol><li>准备管含有2至5毫升冷染色缓冲液（磷酸盐缓冲液（PBS）与添加0.2％BSA和5mm葡萄糖）。置于冰上管组织收获前。 </li><li>根据卡尔小鼠适当批准的协议（如CO 2吸入颈椎脱位）。 </li><li> EDTA或肝素血液收集管进入心脏穿刺，为以后的分析，如红细胞压积，网织红细胞计数或CBC分析，绘制血。 </li><li>收获脾和骨骼，放置在一个单独的管，准备在第1步，从每个鼠标的组织。易于访问以健脾是从左侧。对于骨髓，收获一个或两个股骨。收获的组织置于冰上。 </li><li>如果需要，权衡脾。小鼠接受红细胞生成应激反应可能表现出显着增加脾脏重量。 </li></ol> 2。脾细胞的制备<ol><li>使用预先蘸3毫升注射器推杆，轻轻一推，通过一个40微米的无菌细胞过滤器（Fisherbrand目录编号22363547或其他）脾，或脾的一部分（理想情况下，0.1克或更少，相当于约10 8个细胞）放在一个50毫升的锥形管的顶部。在此过程中试管置于冰上。共2毫升染色缓冲细胞通过过滤网清洗。 </li><li>轻轻吸管紧张的细胞悬液，打破了任何小团块。如果有必要，重新株的细胞。 </li><li>清洗细胞两次离心，重新暂停在寒冷的缓冲区。 </li><li>使用血球计数细胞。典型的产量是1-2 x 10 8细胞/脾脏。流式细胞仪检测分析，aliquote 1至2亿个细胞，每个样品，到流式细胞仪染色管（BD猎鹰聚苯乙烯圆底管，352008）或U型底96孔板（屋宇署猎鹰353910）。样品染色体积为200μL，最后的细胞浓度为0.5〜1 × 10 7细胞/毫升或1-2 × 10 6细胞/ 200μL的样品。 </li></ol> 3。骨髓细胞的制备<ol><li>准备与附加的26G针，预充冷染色缓冲1或3毫升的注射器。 </li><li>取出股骨的肌肉，以可视化骨清楚。 </li><li>使用尖锐的手术剪刀，剪断关闭股骨的两端，尽可能接近骨的两端。这应该揭示了一个小洞，在每个切端，领先进入骨髓腔，通过股骨的长度，运行。 </li><li>在第1步中使用的预充式注射器，通过这些孔插入针，轻轻冲洗骨髓通过小孔在另一端，成筒，。 </li><li>通过上述脾为40微米的过滤器（参见2.1节），游离轻柔吹打刷新细胞和应变。 </li><li>在寒冷的染色缓冲液离心洗涤细胞两次</li><li>计数细胞，重悬在2.4节。典型的产量大约为10 7％，股骨细胞。 </li></ol> 4。胎肝细胞的制备<ol><li>要准备定时怀孕的雌性小鼠，设置在晚上的交配小鼠;检查阴道塞前翌日上午10时，检测阴道塞的一天是一天0.5。兽医人员可能能够帮助那些不熟悉这种技术，以确定怀孕女性的调查。怀孕也可以由监控鼠标重量。 </li><li>定时怀孕的雌性小鼠扑杀12.5至14.5怀孕天。子宫角的Petri盘成含有冰冷的培养基或染色缓冲中删除。 </li><li>胚胎从每个子宫和胎肝解剖。在解剖显微镜所需的胚胎12.5天（E12.5）或以下。 </li><li>可能是肝脏分离机械吹打在缓冲区，并单独处理，在96孔板，或汇集起来，根据实验要求。 </li><li>在E13.5胎肝〜10 7细胞。细胞染色缓冲洗两次，并在1-2 x 10 6细胞/流式细胞仪分析200μL样品重悬于。 </li></ol> 5。抗体染色流式细胞仪<ol><li>准备主抗体染色，预混合被用于所有的样品，对照样品除外，包含以下内容: <ul><li> ChromePure兔IgG（杰克逊015-000-003），到终浓度为200μg/ml。检查瓶子上的股权集中度（它可以改变）。这是用来阻止老鼠细胞的Fc受体，这可能用于替代物种鼠IgG或鼠IgG。树种的选择是由在对大鼠原代/鼠标/兔抗体，这些物种在这种情况下，不能使用导演的二次抗体染色协议中可能存在d为封闭抗体。另外，也可能被用来5％兔血清中纯化IgG的地方。另一种替代方法是使用鼠标Fc受体的单克隆抗体或Fab片段。下面的基本协议不包括任何辅助抗体等可用于任何种抗体。 </li><li> CD71 - FITC，稀释1:200（0.5mg/ml的股票，BD生物科学，553266） </li><li> Ter119 - PE，稀释1:200（股票0.2mg/ml，BD Biosciences公司，553673） </li><li>任何额外的利益表面抗原决定簇的抗体，如抗体冲着FAS或FasL的（见1,2 ）。混合抗体溶液轻轻颠倒离心管2-3次。 </li></ul></li><li>每个细胞样品加入200μL预混合，轻轻地重新悬浮细胞。 </li><li>准备控制细胞样本如下: <ul><li> "不染":留在这些细胞染色缓冲，并提供背景的自发荧光的细胞。 </li><li> "单色"的控制:一个这样的控制是需要为每个抗体/在协议中使用的颜色。这些控件的细胞可以直接标记的抗体染色，或与主抗体和标记的二抗。这些控制是使用正确的渠道之间的光谱重叠。 </li><li> "荧光减一"（FMO）的控制:一个这样的控制是需要在协议中，每个抗体/颜色:细胞除在协议中的所有颜色/抗体染色的颜色/这是FMO控制的抗体。 FMO控制特定通道，该通道的真实背景。它可能包括同一亚型的非特异性抗体，并与测试抗​​体（亚型控制）相同的荧光标记共轭。 </li></ul></li><li> 45&#39;至1小时，在黑暗中（冰水桶放在铝箔盖）在冰上孵育和有关管制的主要抗体染色。 </li><li>孵化结束，洗细胞染色缓冲3毫升3&#39;到5&#39;加入到每个样品管和旋转约400 XG在4 ° C如果使用96孔板，细胞体积200μL洗三次。 </li><li>如果相关，申请二级抗体染色。应用为第一抗体染色洗。 </li><li>如果相关，与Annexin V的染色适用于孵化年底，制造商的指示使用一个HEPES缓冲。这个污点是适用于在室温15分钟，或在冰1小时。 </li><li>细胞重新悬浮，流式细胞仪检测分析中包含一个细胞防渗的DNA染料染色溶液，排除死细胞。一些DNA的染料都可用，包括碘化丙啶，7 - 氨基放线菌素D（7AAD），或DAPI。在这些选择取决于可用的通道特异性抗体染色渠道，和渠道流流式细胞仪。 7AAD是从BD Biosciences公司（559925），并根据制造商的指示使用。 DAPI染色，在二甲基甲酰胺（DMF）为1mg/ml的股票从粉（罗氏​​，CAT＃236276），保持在-20 ° C，染色缓冲液稀释1:10,000至1:15,000。 </li></ol> 6。流流式细胞仪排序<ol><li>细胞对CD71抗体，Ter119，宗族标记，和生存能力染料流流式细胞仪分析（第5）所述的标记。在标签的细胞浓度可提高到5 × 10 7 / ml的。 </li><li>混合同等体积的每个下面的抗体，使宗族主结构: <ul><li> FITC标记的大鼠抗小鼠CD41 MWReg30，BD Pharmingen公司553848 </li><li> FITC标记的鼠抗小鼠CD45R/B220，RA3 - 6B2，BD Pharmingen公司553087 </li><li> FITC标记的鼠抗鼠CD3e 145 - 2C11，BD Pharmingen公司553061 </li><li> FITC标记的鼠抗鼠CD11b/Mac-1 M1/70，BD Pharmingen公司557396 </li><li> FITC标记的鼠抗鼠LY - 6G和Ly - 6C（GR - 1）的RB6 - 8C5，BD Pharmingen公司553126 </li><li> 1:80主混合使用（这相当于每个人的抗体的股票，这是所有0.5毫克/毫升，以1:400稀释）。 </li></ul></li><li>使用低压排序条件和广泛的喷嘴。对于咏叹调（BD Biosciences公司），我们用100μ喷嘴，20 psi的压力。 </li><li>收集缓冲液:PBS与添加20％胎牛血清。 </li><li>要检查的排序种群的纯度，重新从每个样品运行在一个缓冲区，它包含一个可行性的染料（7AAD或类似）一个小等份。 </li></ol> 7。代表性的成果: CD71/Ter119成人骨髓或脾脏染色确定了四个亚群的发展序列，标记PROE，尔雅，EryB EryC（图1）1 。形态，这些对应的日益成熟的红细胞。图1说明了浇注序列数据分析阶段，丢弃非常小的事件（包括细胞核，红细胞），汇总的细胞和死细胞。 可同时测量细胞表面蛋白的表达，每个子集，加入有关的抗体，同时Ter119和CD71染色。图1显示了一个细胞表面死亡受体Fas表达的例子。这种测量方法是在与欧洲专利局注入小鼠进行，或在对照组小鼠注射用生理盐水。很明显，EPO抑制Fas表达在体内 1尔雅人口。 细胞内蛋白质或细胞周期状态的表达，也可以测量细胞在每个子集。图2说明了代表刚收获的骨髓细胞的细胞周期分析。这些测量需要，除了细胞表面染色与CD71和Ter119，细胞细胞内标签（见讨论部分）的固定和通透。 在胎儿肝脏中，首先排除非红系细胞通过门控"林"的细胞，CD41负，MAC - 1，GR - 1，B220和CD3（图3）。其余细胞分分为6个亚群，S0到S5。胎儿肝脏中的细胞的精确模式是依赖于胚胎年龄（见讨论部分）。在E13.5胎肝S3子集的一个有代表性的细胞周期分析显示（图4）。 <img src="/files/ftp_upload/2809/2809fig1.jpg" alt="图1"/> 图1 CD71/Ter119红子集A.浇注战略: 在小鼠脾脏。脾细胞处理和标记CD71，Ter119和Fas抗体。此图显示了以下的数据采集步骤的分析策略。直方图我显示了所有收购的事件。对角线门事件，可能是单细胞，但不包括双峰或更大的聚合。这门细胞直方图二是进一步分析。这里非常小的事件，可能的核或碎片，被排除在外。门控单元格显示直方图三，其中的DAPI阳性细胞，有可能半透膜细胞凋亡，进一步的分析排除。直方图四显示可行的脾细胞产生的人口。 PROE门包含CD71 高 Ter119 中间细胞。 Ter119 高细胞进一步分析直方图五高在这里CD71细胞细分成不太成熟，大的"尔雅"的红细胞（CD71高 Ter119高FSC的高）和更小，更成熟的"EryB" 红细胞（CD71高Ter119 高 FSC的低） 。最成熟的红细胞的一个子集EryC（CD71低Ter119高FSC 的低） 。直方图VI，显示细胞表面Fas表达，特别是在尔雅子集，在基础状态下的小鼠（注射用生理盐水），并与单剂量注射的EPO的老鼠，。 Fas抗体染色同时CD71/Ter119染色。Cytospin细胞的每一个指定的子集进行排序的准备。细胞与姬姆萨染色，并与二氨基联苯胺，后者与血红蛋白生成褐色的污渍。刘等人最初发表Cytospin数据，血。 2006年7月1 108（1）:123 - 33。 EPUB 2006年3月9日。 <img src="/files/ftp_upload/2809/2809fig2.jpg" alt="图2"/> 图2 CD71 高 Ter119在小鼠骨髓红细胞的细胞周期分析 。小鼠注射用BrdU腹腔，脾或骨髓收获30至60分钟后。细胞固定和通透，除了被CD71和Ter119染色，染色BrdU掺入到他们的DNA复制与BrdU定向单克隆抗体（固定，通透性和BrdU染色协议是根据制造商的指示）。 BrdU阳性细胞周期S期。间期细胞BrdU阴性和可能解决的G1或G2 / M期，使用DNA染料7AAD。 <img src="/files/ftp_upload/2809/2809fig3.jpg" alt="图3"/> 图3 CD71/Ter119红子集A.浇注战略: 在小鼠胎肝。胎肝细胞标记CD71，Ter119，并在非红系标记（"林"）执导的FITC标记的抗体鸡尾酒。林表达活菌（7AAD阴性）进行了分析，和林细胞进一步细分到S0到S5红子集。雅戈尔，是由E13胎肝细胞在成熟的S4/S5子集的情况下表现出不太成熟的红细胞，从每一个指定的子集进行排序的细胞B. Cytospin的准备工作。细胞与姬姆萨染色，并与二氨基联苯胺，后者与血红蛋白生成褐色的污渍。 Cytospin数据最初published公共科学图书馆生物学流行等 ，8（9）:e1000484。作者:10.1371/journal.pbio.1000484。 <img src="/files/ftp_upload/2809/2809fig4.jpg" alt="图4"/> 图4。胎肝红亚群细胞周期分析。孕鼠注射用BrdU，胎儿肝脏收获30至60分钟后，固定，透，并与抗体对CD71，Ter119和BrdU染色。 S3细胞的细胞周期的状态显示。

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动物实验是由马萨诸塞州医学院IACUC委员会大学规定的准则和法规的规定执行。

流流式细胞仪的方法允许同时调查任何可能与一个特定的荧光标记抗体或配体，包括细胞表面标志物，蛋白表达，细胞的存活，使用磷酸化特异性抗体和细胞周期状态的细胞信号检测的细胞功能。这些测量可以在每一个分化阶段的特定子集，在新鲜分离的红细胞生成组织范围内。因此，这种方法允许红细胞生成系统的不同层次的功能和分子的变化进行评估，在广泛的红细胞生成刺激或基因突变的?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> FAS -生物素</td><td> BD Pharmingen公司</td><td> 554256 </td></tr><tr><td>链霉亲和APC </td><td>分子探针</td><td> S868 </td></tr><tr><td> 40微米的无菌细胞过滤器</td><td> Fisherbrand </td><td> 22363547 </td></tr><tr><td>流式细胞仪染色聚苯乙烯圆底管</td><td>屋宇署猎鹰</td><td> 352008 </td></tr><tr><td> U型底96孔板</td><td>屋宇署猎鹰</td><td> 353910 </td></tr><tr><td> ChromePure兔IgG </td><td>杰克逊ImmunoResearch </td><td> 015-000-003 </td></tr><tr><td> CD71 - FITC（股票0.5mg/ml的） </td><td> BD -生物科学</td><td> 553266 </td></tr><tr><td> Ter119 - PE（股票0.2mg/ml） </td><td> BD -生物科学</td><td> 553673 </td></tr><tr><td> 7AAD </td><td> BD -生物科学</td><td> 559925 </td></tr><tr><td> DAPI染色粉</td><td>罗氏公司</td><td> 236276 </td></tr><tr><td> FITC标记的大鼠抗小鼠CD41 MWReg30 </td><td> BD Pharmingen公司</td><td> 553848 </td></tr><tr><td> FITC标记的大鼠抗小鼠CD45R/B220，RA3 - 6B2 </td><td> BD Pharmingen公司</td><td> 553087 </td></tr><tr><td> FITC标记的大鼠抗小鼠CD411b/Mac-1 M1/70 </td><td> BD Pharmingen公司</td><td> 557396 </td></tr><tr><td> FITC标记的鼠抗鼠LY - 6G和Ly - 6C（GR - 1）RB6 - 8C5 </td><td> BD Pharmingen公司</td><td> 553126 </td></tr><tr><td> FITC标记的鼠抗鼠CD3e 145 - 2C11 </td><td> BD Pharmingen公司</td><td> 553061 </td></tr><tr><td> APC BrdU流试剂盒</td><td> BD Pharmingen公司</td><td> 557892 </td></tr><tr><td> Annexin V的抗生物素蛋白</td><td> BD Pharmingen公司</td><td> 556418 </td></tr></tbody></table>

identification and analysis of mouse erythroid progenitors using the cd71/ter119 flow-cytometric assay

红细胞的研究，旨在了解红细胞是如何从早期的造血和红系祖细胞形成。具体来说，是受激素的促红细胞生成素（EPO），其合成是由组织缺氧引起红细胞的形成率。适当组织在迅速增加EPO的充氧效果，驾驶增加红细胞生成率的威胁，被称为红细胞生成应激反应的一个过程。在循环红细胞的数量增加，提高组织的氧输送。红细胞生成一个高效的应激反应，因此，从生理和病理条件下，如高海拔，贫血，出血，化疗或造血干细胞移植的生存和恢复的关键。 鼠标是红细胞和应激反应的研究重点型号。鼠标最终（成人型）红细胞在胚胎12.5和15.5之间天的胎肝，脾中的新生儿，在成人脾和骨髓。确定组织中的红系祖细胞的经典方法依赖于这些细胞的能力，引起红细胞殖民地时，在半固体培养基含有EPO -镀。他们的红易制毒化学后代基于形态学标准确定。无论这些经典的方法能够获得大量的细胞分子研究的特定分化阶段红。在这里，我们目前的流量识别和研究直接在新鲜分离的小鼠组织中的分化阶段特有的红系祖细胞和前体，流式细胞仪方法。检测依赖于细胞表面标志物CD71，Ter119，流流式细胞仪"向前散射"的参数，这是一个细胞大小的功能。 CD71/Ter119法可用于研究红系祖细胞在其体内红细胞生成应力的响应，例如，住在贫血小鼠或老鼠在低氧条件。直接转基因的成年小鼠或胚胎组织，它也可以用来研究红系祖细胞，修改后的红细胞中的分子途径，以评估的具体作用。

Watch this Scientific Journal Video about Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay at JoVE.com

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

一个流式细胞仪方法，鉴定和分化阶段特异性的小鼠红系祖细胞和前体，直接在新鲜收获的小鼠骨髓，脾脏或胎肝的分子分析流。检测依赖于细胞表面标志物CD71，Ter119，细胞大小。

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

identification-analysis-mouse-erythroid-progenitors-using-cd71ter119

Research

JoVE Journal

Biology

31.9K 次观看.  University of Massachusetts Medical School. 一个流式细胞仪方法，鉴定和分化阶段特异性的小鼠红系祖细胞和前体，直接在新鲜收获的小鼠骨髓，脾脏或胎肝的分子分析流。检测依赖于细胞表面标志物CD71，Ter119，细胞大小。

视频: Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

科研

生物学

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

双分子荧光互补流式细胞仪分析:高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

细胞调理介质细胞外囊泡的流式细胞学分析

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

大鼠骨骼肌中纤维-脂肪源性祖细胞（FAPs）和肌源性祖细胞（MPs）的鉴定、分离和表征

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

小鼠巨核细胞祖细胞的分离

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...

小鼠胰腺 β 细胞中线粒体自噬的定量分析

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

作者聚焦:使用有效且稳健的互补方法评估胰腺 β 细胞中的线粒体自噬通量 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

研究胰腺β细胞线粒体自噬通量的互补方法

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

使用成像流式细胞术定量分析微生物自聚集

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

作者聚焦：使用成像流式细胞术推进微生物自聚集的研究

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

成像流式细胞术研究微生物自聚集

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...