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。

<ol>
	<li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+Fas-FasL+coexpression+by+erythropoietin+mediates+erythroblast+expansion+during+the+erythropoietic+stress+response+in.">Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in.</a> 108, 123-133 (2006).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Autoregulation+by+FAS+Mediates+Robust+Fetal+Erythropoiesis.">Negative Autoregulation by FAS Mediates Robust Fetal Erythropoiesis.</a> PLoS Biol. 5, e252-e252 (2007).</li><li>Krutzik, P. O., Hale, M. B., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+murine+immunological+signaling+network+with+phosphospecific+flow+cytometry.">Characterization of the murine immunological signaling network with phosphospecific flow cytometry.</a> J Immunol. 175, 2366-2373 (2005).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ineffective+erythropoiesis+in+Stat5a(-/-)5b(-/-)+mice+due+to+decreased+survival+of+early+erythroblasts.">Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.</a> Blood. 98, 3261-3273 (2001).</li><li>Guihard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAPK+ERK1+is+a+negative+regulator+of+the+adult+steady-state+splenic+erythropoiesis.">The MAPK ERK1 is a negative regulator of the adult steady-state splenic erythropoiesis.</a> Blood. 115, 3686-3694 (2010).</li><li>Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+erythroid+chaperone+that+facilitates+folding+of+alpha-globin+subunits+for+hemoglobin+synthesis.">An erythroid chaperone that facilitates folding of alpha-globin subunits for hemoglobin synthesis.</a> J Clin Invest. 117, 1856-1865 (2007).</li><li>Chen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythroid+dysplasia,+megaloblastic+anemia,+and+impaired+lymphopoiesis+arising+from+mitochondrial+dysfunction.">Erythroid dysplasia, megaloblastic anemia, and impaired lymphopoiesis arising from mitochondrial dysfunction.</a> Blood. 114, 4045-4053 (2009).</li><li>Chen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+distinct+stages+in+erythroid+differentiation+based+on+dynamic+changes+in+membrane+protein+expression+during+erythropoiesis.">Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.</a> Proc Natl Acad Sci U S A. 106, 17413-17418 (2009).</li><li>McGrath, K. E., Bushnell, T. P., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+hematopoietic+cells:+where+flow+meets+morphology.">Multispectral imaging of hematopoietic cells: where flow meets morphology.</a> J Immunol Methods. 336, 91-97 (2008).</li><li>Pop, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+key+commitment+step+in+erythropoiesis+is+synchronized+with+the+cell+cycle+clock+through+mutual+inhibition+between+PU.1+and+S-phase+progression.">A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression.</a> PLoS Biol. 8, (2010).</li><li>Borsook, H., Lingrel, J. B., Scaro, J. L., Millette, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+haemoglobin+in+relation+to+the+maturation+of+erythroid+cells.">Synthesis of haemoglobin in relation to the maturation of erythroid cells.</a> Nature. 196, 347-350 (1962).</li></ol>

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结果。 没有抗体无交叉反应，交叉反应可能是组织特异性。来验证，因此重要的是，即使是以前测试的抗体，其特异性红细胞生成组织的背景下，使用一个空或敲除的细胞模型。 从具体的红亚群的细胞可能是排序或RNA分子在转录组学分析。排序实验应​​使用低排序的压力和广泛的喷嘴，以尽量减少对细胞的剪切应力。我们建议您检查每个分拣实验后细胞纯度和活力。值得注意的是，在多参数流式细胞仪检测的情况下，每种颜色的真实背景是"鱼类统营处"控制（见5.3），其中包括，除了自体荧光，由于所有其他颜色的光谱重叠的背景。在分析阶段，需要使用特定的一个子集的"鱼类统营处"控制。 分化阶段的红细胞内的流动cytometrically定义的子集流流式细胞仪PROE /尔雅/ EryB / EryC亚群定义在细胞表面标志物表达和前向散射。虽然它很可能，这些子集对应于在多种小鼠模型大致相同的形态红细胞分化阶段，我们建议您验证这个新的小鼠模型研究。应进行排序，每个流cytometrically定义的亚群细胞和红细胞形态分期审查cytospin的准备工作。 虽然CD71/Ter119子集都包含类似的分化阶段的红细胞，仍然存在的异质性程度在每个子集。在第一个应用程序的CD71/Ter119方法，我们分为I区CD71表达对他们的基础第四Ter119 +细胞。这些地区之间的确切边界确定任意4。我们随后补充说:细胞大小的信息分析，前向散射参数的形式。这让我们分为以下人口自然轮廓（图1）子集Ter119高细胞。这种做法导致更均匀成熟的人群，更可重复性的结果， 由 5,6,7其他研究人员最近已适应。尔雅的子集可能需要的地方7分，被进一步划分。一组建议使用CD44 CD71 8。虽然CD44是在解决早期红细胞阶段用处不大，它可能会更精确地解决以后的红细胞亚群。两个标记，因此可能被使用，或者，他们的选择可能取决于利益的特定子集。 另一种策略，采用多光谱成像使用ImageStream技术（Amnis公司，西雅图，WA）9 。它使成千上万的细胞形态和流流式细胞仪数据的同步和快速采集。它很可能成为"金标准"的特定阶段，红细胞的分子分析方面，自分化阶段定义的形态学标准，可直接测量。然而，在目前这种技术比传统的流式细胞仪广泛，并受到两个缺点:它不允许细胞分选;它仅限于数量较少的流动流式细胞仪参数。 细胞内的抗原检测细胞内蛋白质或BrdU的需要细胞固定和通透。精确的固定和通透的过程取决于在细胞内抗原的问题。我们在固定过程中使用的活/死可以解决的死细胞染色（分子探针），以区别于可行的死细胞。值得注意的是，用清洁剂的通透性通常影响，这是部分洗涤剂溶于Ter119信号。使用温和的洗涤剂（如皂甙"烫发/洗"BD公司的缓冲区），我们克服了这一困难。我们也染色Ter119两个前，下面，固定和通透的程序，以优化Ter119 SIGNAL。另外，它是可能的排序从每个CD71 / Ter119子集第一可行的细胞，并进行固定和通透性，分别从每个子集（例如，看到在胎肝细胞周期分析 ）10纯化细胞。 胎肝CD71/Ter119亚群在胎儿肝脏CD71/Ter119染色模式是依赖于胚胎2岁（图3）。我们划分为6个亚群，S0到S5 10胎肝细胞。细胞在胚胎第11天（E11）最终红细胞的胎肝的发病，主要集中在亚群S0和S1，主要是红系集落形成细胞（CFU - E）。随着胚胎发育，CFU - E细胞分化成proerythroblasts和成熟的红细胞，并逐步填充的子集S2 S5（图3）。 子集S1至S5是几乎完全明确的谱系红系细胞组成。这些子集EPOR - / -胎肝中缺席。一个Ter119 +细胞在胎儿肝脏中的少数对应的原始（卵黄囊）红系。这些细胞是显而易见的EPOR - / -胎肝，没有明确的谱系红细胞产生，而是由E13.5形式小于1 Ter119 +细胞在野生型胎儿肝脏 10％ 。 S0的子集是异类。在E13.5，S0的细胞中70％是红系细胞，CFU - E的阶段，只是前10 EPOR依赖发病。其余的早期祖细胞以及其他造血谱系，主要是巨核细胞和巨噬细胞，这些细胞可能是排序或出 10门（图3） 。 红子集的频率变化的释义红子集的频率的变化，应小心解释。在任何给定的子集细胞频率的变化可能是由于他们改变的细胞凋亡率，通过该子集过境时间改变，或者在其他亚群中的细胞数量的变化可能是由于。早期红细胞亚群PROE和尔雅的次数增多的一个常见的原因是多重病因1红细胞生成应力。类似的调查结果已经注意到在1960年的红细胞形态检查过程中的应激反应11。在早期前体的相对频率，在压力增加的确切原因尚不清楚，但部分可能是由于这些前体在改善生存压力1。

<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

Nanyang Technological University

Research

JoVE Journal

Biology

32.0K 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 Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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 ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

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.

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 and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

各种鼠标脂肪库的识别和解剖

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...