致谢研究流式细胞仪核心在辛辛那提儿童医院研究基金会和理查德德马科，Sherree朋友，和斯科特末底改从Amnis公司（EMD Milllipore的一部分）的专家技术支持。这项工作是由美国国立卫生研究院赞助授予K08HL088126和R01HL116352（TAK）和P30 DK090971（YZ）。

1，长期体外培养红细胞生成（ 体外红系分化培养议定书Giarratana 等。8，修改和调整鼠标电池） 这是一个三步骤的长期体外红细胞生成协议。在第一步骤（天0-4）2×10 5个细胞/ ml被放置在补充有干细胞因子（SCF），白细胞介素-3（IL-3），和促红细胞生成素（EPO）的红细胞的生长培养基。在第二步骤（天5-6），将细胞重新悬浮于2×10 5个细胞/ ml和共培养的贴壁基质细胞（MS5）在新鲜红细胞生长培养基中只用Epo的补充。在第三步骤（天7-9），细胞是在一层的MS-5细胞在新鲜红细胞生长培养基的无细胞因子高达摘除术（ 图1A）中培养。 所有动物的协议已获的机构动物护理和使用委员会（IACUC）辛辛那提儿童医院医疗中心。 <ol><li>骨骼和低密度的骨髓细胞中分离的收获<ol><li>加入2毫升含在一个15毫升锥形管2％胎牛血清（FBS）的无菌IMDM培养，并保持在冰上。 </li><li>安乐死2-6个月大的野生型C57/BL6小鼠（连同或不带基因靶向小鼠的利息）以下机构批准的协议（ 如 CO 2吸入，颈椎脱位）。 </li><li>隔离骨盆骨，股骨，和两条腿的胫骨用钳和手术刀，将它们添加到含IMDM +2％FBS管并保持在冰上。 </li><li>加入1 ml IMDM 2％FBS的使用镊子和结核菌素注射器用25克X穿过骨头5/8“针。冲洗IMDM 2％FBS的几次轻轻无菌流式细胞仪管和冲洗骨（通过吸〜500微升的细胞悬浮液，并通过骨再冲洗的话），并收集骨髓细胞进入流cytometrŸ管。法拉盛是完整的骨骼时显示为白色。 </li><li>通过在50毫升锥形管的顶部有40微米的细胞过滤设置过滤细胞悬液。用IMDM 2％FBS中，并使用相同的培养基调整细胞悬浮液的最终体积至5ml洗细胞滤网。 </li><li>通过密度梯度离心制备的低密度的骨髓细胞（LDBM）：仔细层5毫升细胞悬液上5毫升密度梯度分离细胞的培养基1.083在15-ml管中并旋转，在750×g离心25微克/毫升分钟，在室温（RT）与不加制动/低加速度。 </li><li>转移上清液（一切高达约2毫升标记的15-ml的试管中，以获得血沉棕黄层）到一个新的15毫升管中并持续5分钟，在执行多一个洗涤用IMDM 2％FBS中于525×g离心RT。 </li><li>吸出上清液，并通过悬浮颗粒在3ml红细胞裂解缓冲液中5分钟，在室温下溶解任何残留的红血细胞（RBC）。如果红细胞的更大的纯度是在（ 例如用于生化研究）的最后一步需要，进一步净化LDBM细胞这一步骤林负细胞磁分离。 </li></ol></li><li> 体外红系分化培养协议，从LDBM或林NEG细胞开始（ 图1A） <ol><li>加7毫升IMDM 2％FBS中，自旋在525×g离心5分钟，在室温下，重悬沉淀的细胞在2ml红细胞生长培养基（EGM），其中包括： 一个。 STEMPRO-34培养基中，含 湾2.6％STEMPRO-34中型补充， 角20％BIT-9500， ð。 900毫微克/毫升的硫酸亚铁， Ë。 90纳克/毫升的硝酸铁， f。 100单位/ ml青霉素/链霉素，2mM的L-谷氨酰胺 克10 -6 M氢化可的松 小时。而新加入的细胞因子： I）100毫微克/毫升的SCF， ⅱ）5毫微克/毫升IL-3，并 ⅲ）3 IU / ml的促红细胞生成素。 </li><li>使用自动细胞计数器或手动在显微镜使用下摆细胞计数ocytometer。 </li><li>板在6孔细胞培养板中以5×10 5细胞/孔，以2.5毫升在EGM（2×10 5 LDBM细胞/ ml）的最终体积和孵育在37℃下的浓度（这被认为是如白昼＃文化0）。 </li><li>通过吸1.5毫升上清液，纺纱在525×g离心5分钟，在室温下，再悬浮于1.5ml含有所有3种细胞因子和天＃2加回至孔中每天新鲜培养基更换培养基，3，4，以优化增生期。在第3天，取出所有的细胞，计数和分裂成井的适当数量，以维持的〜2×10 5细胞/ ml和细胞浓度。保持文化在37℃/ 5％的CO 2。 </li><li>在第4天，板MS5细胞（小鼠基质细胞系），以一个新的细胞培养6孔板的孔中。该MS5细胞培养基α-MEM中含有20％FBS，100单位/ ml青霉素/链霉素和2mM L-谷氨酰胺。 </li><li>第5天，数数细胞（现在在红细胞显著富集）在原始培养板用自动细胞计数器或手动在使用血细胞计数显微镜各孔中。 </li><li>解除所有细胞从每个孔中，转移到15毫升锥形管，降速在525×g离心5分钟，在室温，吸出上清液，并溶解粒或仅含有促红细胞生成素（3单位/毫升）以2×10的浓度新鲜EGM 5细胞/ ml。 </li><li>从其中的MS-5细胞的前一天（针对70-80％汇合在共培养时）镀和添加的类红细胞在这些井在EGM仅含有促红细胞生成素的孔中吸出上清液（虽然不是他们的介质的选择MS-5细胞存活以及在股东特别大会上数天）。 </li><li>改变介质上的一天＃6加入新鲜的EPO。 </li><li>在一天＃7更改介质，采用临时股东大会没有添加细胞因子。第7天到第9，测试样本为眼球摘除与流量具有抗TER119和祥发，16日及P后流式细胞仪roceed到染色样品的多光谱成像流式细胞术（如第3节详述）。 注意：电镀前和第2天，4，6，和文化7，流式细胞仪通过评估表面标志CD44和TER119与尺寸（FSC）监测培养细胞为红细胞富集和分化与流动9或者CD71，TER119，和FSC也可以用10，11。 </li></ol></li></ol> 2，快速摘除术含量，据Yoshida 等人描述的协议。12与修改（图1B） <ol><li>应激诱导红细胞生成和间质细胞制备方法在体外培养红细胞生成<ol><li>每麻醉IACUC准则2-6个月大的野生型C57/BL6小鼠（ 如异氟醚溶液）。确保鼠标已充分通过检查无反射性反应，以温和的后腿爪子捏和麻醉在手术过程中经常呼吸。使用上的眼睛兽医眼药膏，以防止干燥时在麻醉下。 </li><li>通过尾部出血的500微升的最终体积诱发应激红细胞生成。使用胰岛素注射器，注射生理盐水等体积腹腔注射，以保证动物的液体复苏（保持在头低脚高位动物注射前和注射于下腹部，以免损伤内脏）。 </li><li>请勿将鼠标无人值守，直到它重新获得电机控制的动物开始走动笼，并能够站立和行走没有掉下来表示。该phlebotomized鼠标放置在笼中没有其他小鼠。 </li><li>术后第2天，板MS-5细胞在24孔细胞培养板的孔。孵育MS-5细胞在37℃/ 5％CO 2中MS5细胞培养基（含有20％FBS的α-MEM，100单位/ ml青霉素/链霉素和2mM L-谷氨酰胺），其目的是为70-80 ％铺满我48小时在n板孔。 </li></ol></li><li>脾和脾细胞处理的收获<ol><li>应力诱导红细胞生成四天（96小时）后，通过安乐死IACUC批准的协议（ 如 CO 2吸入颈椎脱位）以前流血鼠标。 </li><li>收获脾，把它放在一个15毫升的锥形管的IMDM含2％FBS和保持在冰上。 </li><li>早在实验室里，在无菌条件下组织培养罩，反转含脾管上40微米的细胞过滤在50毫升管的顶部设置。用5毫升的塑料注射器的柱塞挤压脾。 </li><li>用IMDM 2％FBS冲洗细胞过滤器，并使用相同的培养基，调整细胞悬浮液的最终体积至5ml。 </li><li>仔细层上5毫升密度梯度分离细胞的5毫升脾细胞悬浮液介质1.083克/ ml的15毫升管中，自旋，在750×g下在室温下25分钟内，没有制动/低加速度。 </li><li>转让上清液（溶液至约2毫升标记的15-ml的试管中，以获得血沉棕黄层）到一个新的15毫升管中，并执行多一个洗涤用IMDM 2％FBS中于525×g离心5分钟，在室温。 </li><li>吸出上清液，并裂解红血细胞悬浮的沉淀物在3ml红细胞裂解缓冲液中5分钟，在RT。 </li><li>加7毫升IMDM 2％FBS冲洗细胞，并稀释和中和RBC裂解缓冲液和旋在525×g离心5分钟，在4℃下</li><li>吸出上清液，并暂停沉淀的细胞在2毫升红细胞生长培养基（EGM）的。 </li></ol></li><li>孤立的低密度脾，富含红细胞，塑料（快速体外培养红细胞生成的第一阶段）文化<ol><li>依靠自动细胞计数器或手动血球细胞。细胞中分离每脾在这个阶段的通常数量为〜15×10 6。 </li><li>暂停细胞进一步在EGM含有细胞因子（在fINAL浓度）：SCF 50 ng / ml的IL-3 5毫微克/毫升，并生成素2单位/毫升。 </li><li>板1-5×10 6个细胞在24孔细胞培养板的1毫升/井（同每孔取决于细胞总数数量的细胞）的最终体积，并培育O / N在37℃/ 5％的CO 2。 </li></ol></li><li>对MS5细胞红细胞（快速体外培养红细胞生成第二阶段的文化<ol><li>从塑料以及抽吸上清液，并加入2毫升冷的10mM EDTA的PBS中5分钟，在冰上到每个孔中抬起细胞（高度浓缩的红细胞）。 </li><li>把细胞在新管，在股东特别大会上洗一次（无细胞因子），并悬浮在相同的。 </li><li>板5×10 5 -1×10 6个细胞在1-2毫升的量到各MS5细胞涂于24孔板中的井。如果药理抑制剂在实验中被使用，现在加上他们在适当的浓度。 </li><li>孵育6〜8小时（时间由导游显微镜观察未处理的野生型样品中约30％-40％去核）的。红细胞的到MS-5细胞的结合加速其摘除术。 </li><li>通过加入2ml冷PBS +的10mM的EDTA，5分钟，在冰上从每个孔中抬起细胞。随着红细胞，MS-5细胞也将被收集，但这些可以稍后在流式细胞仪分析FSC 喜 TER119轻易排除-细胞。 </li><li>在这个阶段中，细胞可以是固定的和染色的由多光谱成像流式细胞仪分析。 </li></ol></li></ol> 3，红细胞的染色对细胞内蛋白质或脂筏的本地化过程中摘除由多光谱成像流式细胞仪<ol><li>在PBS中洗涤细胞，旋转525×g离心5分钟，在RT和吸出上清液。 </li><li>通过重悬细胞沉淀中加入500μl在PBS中的3.7％甲醛15分钟，固定细胞（取决于抗固定的时间可能会发生变化根被探测），并轻轻吹打。 </li><li>转移到1.5毫升的塑料离心管中并孵育15分钟，在RT。 </li><li>旋转的长凳上，离心2000×g离心20秒，吸出上清液，加入500微升PBS以每管和移液轻轻地执行一个洗。 </li><li>旋转的长凳上，离心2000×g离心20秒，吸出上清液并保持在冰上管至少15分钟。 Permeablization步骤3.6-3.9应迅速和有效地完成，并在室温下旋转/抽吸，细胞沉淀应立即放回冰，为了使细胞更好地保持其完整性。 </li><li>取丙酮溶液从-20°C冰箱装上冰水。在500微升冰冷的50％丙酮（1:1 DH 2 O）和移液轻轻重悬首细胞沉淀通透细胞。 </li><li>旋上板凳离心2,000 xg下在500微升20秒，吸出上清，重悬细胞沉淀60，冰冷的100％丙酮轻轻吹打。 </li><li>旋上板凳离心2000×g离心20秒，吸出上清，重悬细胞沉淀再一次在500微升冰冷的50％丙酮轻轻吹打。 </li><li>旋上板凳离心2000×g离心20秒，吸出上清液，轻轻吹打在寒冷的FACS缓冲液（PBS +0.5％BSA）洗细胞一次。 </li><li>准备贴标鸡尾酒抗体或标记感兴趣的分子：0.1 U/100微升AF488-鬼笔环肽对F-肌动蛋白染色和1μl/100微升TER119-PECy7为红系细胞染色。替代的或附加的染色，也可使用AF-488-抗β-微管蛋白抗体（1:50），AF-594标记的霍乱毒素B亚单位标记脂质筏（1:200），抗pMRLC（Ser19）做对于磷酸化肌球蛋白调节轻链（1:50），其次是抗兔AF-488标记的二抗（1:400）和抗-γ-徒步鞋初级抗体林初级兔抗体（1:100），接着加入抗兔AF-555标记的二抗（1:300）。 </li><li>下面上清吸出，重悬细胞沉淀中加入100μl标记的鸡尾酒，吸管轻轻孵育在室温30分钟。 </li><li>制备含核染色DRAQ5 2.5微米FACS缓冲液中。 </li><li> FACS缓冲液洗涤细胞，降速板凳上的离心2000×g离心20秒，吸出上清，重悬在含有DRAQ5 60微升FACS缓冲液中。 </li><li>在摄像运行样品流式细胞仪收集每个实验至少10,000个事件，补偿的原始数据文件如先前公布的13，和如图2分析结果，使用分析软件特有的成像流式细胞仪。 </li></ol>

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作者宣称没有竞争的财务权益。

近年来红细胞去核的研究已经获得了越来越多的动力，因为它是在步骤在体外红细胞生成培养物是最困难的，以实现成功的，大规模生产的红血细胞在体外有效地重现。直到最近，红细胞去核的研究主要是利用荧光显微镜和流式细胞仪的方法。荧光显微镜的方法，虽然在确定参与分子有帮助的，需要显微镜观察，以确定一个小数目正色红细胞数百个细胞固定于在特定时间点内进行眼?...

在哺乳动物中红细胞系内终末分化的结论与去核的戏剧性的过程，通过该正色红细胞排出其膜包裹的核（pyrenocyte）1，产生一个网织红细胞2。这个过程的确切机制，这也是成功的限速步骤，大规模生产的红血细胞在体外 ，还没有完全阐明。去核红细胞内关键的蛋白质和结构的定位依赖于使用荧光显微镜和电子显微镜3-5。这个繁琐的过程通常会导致的眼球摘除活动数量有限的识别，并且不总是允许有意义的统计分析。扩大对红细胞鉴定麦格拉思等 6前面所述的方法，我们已经开发了一种新的由多光谱伊马确定和研究眼球摘除事件的方法更改流式细胞仪（多参数高速细胞成像中流动，它结合了荧光显微镜与流式细胞术的方法）7，它可以提供的观测足够数量以获得测量值，并执行统计分析。 在这里，我们描述了前两个在体外培养红细胞生成为了同步红细胞和增加捕获眼球摘除在评估时的概率使用的方法。然后，我们详细描述了为了眼球摘除由多光谱成像流式细胞仪中，研究细胞内蛋白质或脂质筏的本地化固定和透后红细胞的染色。 样品上的成像流运行流式细胞仪和将收集的细胞的门控适当地识别正色红细胞6。正色红细胞，然后根据它们的纵横比进行分析，测得在明场图像进行更改，对他们的参数增量的质心的XY TER119-DNA，其被定义为分别染色TER119和DNA的区域中，中心之间的距离值。细胞低高宽比和高三角形质心的XY Ter119/DNA的人口是高度浓缩的去核的细胞。用野生型（WT）与红细胞与红细胞的Rac1蛋白在rac2上的Mx-Cre重组酶介导的条件缺失- / -或组合的Rac2 - / - ; RAC3 - / -遗传背景和多光谱成像流这种新型分析仪的协议，我们最近表明，眼球摘除类似于非对称细胞分裂，并通过外消旋GTP酶在调节部分的肌动球蛋白环的形成是眼球摘除进展7重要。

<table border="0" cellpadding="0" cellspacing="0" width="934">
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		<tr height="22">
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		<tr height="29">
			<td height="29" style="height:29px;width:363px;">IMDM medium</td>
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			<td style="width:199px;">GIBCO (Life Tech)</td>
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		<tr height="30">
			<td height="30" style="height:30px;width:363px;">Stempro-34 nutrient supplement</td>
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		<tr height="24">
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			<td style="width:207px;">512450</td>
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			<td style="width:199px;">Stemcell Technologies</td>
			<td style="width:207px;">09500</td>
			<td style="width:167px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:207px;">BP-1600-100</td>
			<td style="width:167px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:363px;">Phosphate buffered saline (PBS)</td>
			<td style="width:199px;">Hyclone (Thermo Scientific)</td>
			<td style="width:207px;">SH30028.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:363px;">Penicillin/Streptomycin</td>
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			<td style="width:207px;">SV30010</td>
			<td style="width:167px;"></td>
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		<tr height="23">
			<td height="23" style="height:23px;width:363px;">L-glutamine</td>
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			<td style="width:207px;">SH30590.01</td>
			<td style="width:167px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:363px;">Isothesia (Isoflurane)</td>
			<td style="width:199px;">Butler-Schein</td>
			<td style="width:207px;">029405</td>
			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Histopaque 1.083mg/ml</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:207px;">10831</td>
			<td style="width:167px;"></td>
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		<tr height="27">
			<td height="27" style="height:27px;width:363px;">BD Pharmlyse (RBC lysis buffer)</td>
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			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Acetone</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:207px;">534064</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:363px;">Formaldehyde</td>
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			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Hydrocortisone</td>
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			<td style="width:207px;">H4001</td>
			<td style="width:167px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Stem Cell Factor (SCF)</td>
			<td style="width:199px;">Peprotech</td>
			<td style="width:207px;">250-03</td>
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		<tr height="22">
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			<td height="34" style="height:34px;width:363px;">EPOGEN Epoetin Alfa (Erythropoietin, EPO)</td>
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			<td height="31" style="height:31px;width:363px;">CD44-FITC antibody</td>
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		<tr height="28">
			<td height="28" style="height:28px;width:363px;">CD71-FITC antibody</td>
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			<td style="width:207px;">553266</td>
			<td style="width:167px;"></td>
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		<tr height="30">
			<td height="30" style="height:30px;width:363px;">Ter119-PECy7 antibody</td>
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			<td style="width:207px;">557853</td>
			<td style="width:167px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:363px;">Phalloidin-AF488</td>
			<td style="width:199px;">Invitrogen (Life Technologies)</td>
			<td style="width:207px;">A12379</td>
			<td style="width:167px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:363px;">&#946;-tubulin-AF488 antibody</td>
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			<td style="width:207px;">#3623</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">anti-rabbit AF488-secondary antibody</td>
			<td style="width:199px;">Invitrogen (Life Technologies)</td>
			<td style="width:207px;">A11008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">anti-rabbit AF555-secondary antibody</td>
			<td style="width:199px;">Invitrogen (Life Technologies)</td>
			<td style="width:207px;">A21428</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">AF594-cholera toxin B subunit</td>
			<td style="width:199px;">Invitrogen (Life Technologies)</td>
			<td style="width:207px;">C34777</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">pMRLC (Ser19) antibody</td>
			<td style="width:199px;">Cell Signaling</td>
			<td style="width:207px;">#3671</td>
			<td style="width:167px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:363px;">&#947;-tubulin antibody</td>
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			<td style="width:207px;">T-3559</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">Syto16</td>
			<td style="width:199px;">Invitrogen (Life Technologies)</td>
			<td style="width:207px;">S7578</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Draq5</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Ferrous sulfate</td>
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			<td style="width:207px;">F7002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Ferric nitrate</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:207px;">F3002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">EDTA</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:207px;">BP120500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:363px;">15ml tubes</td>
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			<td style="width:207px;">352099</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:363px;">50ml tubes</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:363px;">6-well plates</td>
			<td style="width:199px;">BD Falcon</td>
			<td style="width:207px;">353046</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:363px;">24-well plates</td>
			<td style="width:199px;">BD Falcon</td>
			<td style="width:207px;">351147</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:363px;">Flow tubes</td>
			<td style="width:199px;">BD Falcon</td>
			<td style="width:207px;">352008</td>
			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tuberculin syringe</td>
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		<tr height="22">
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			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Syringe needle 25G5/8</td>
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			<td style="width:207px;">305122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Capped flow tubes</td>
			<td style="width:199px;">BD</td>
			<td style="width:207px;">352058</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:363px;">40&#956;m cell strainer</td>
			<td style="width:199px;">BD Falcon</td>
			<td style="width:207px;">352340</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Scalpel (disposable)</td>
			<td style="width:199px;">Feather</td>
			<td style="width:207px;">2975#21</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">FACS Canto Flow Cytometer</td>
			<td style="width:199px;">BD</td>
			<td style="width:207px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">ImagestreamX Mark II Imaging Flow Cytometer</td>
			<td style="width:199px;">AMNIS (EMD Millipore)</td>
			<td style="width:207px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:363px;">Image Data Exploration and Analysis Software (IDEAS) version 4.0 and up.</td>
			<td style="width:199px;">AMNIS (EMD Millipore)</td>
			<td style="width:207px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:363px;">Hemavet 950 Cell Counter</td>
			<td style="width:199px;">Drew Scientific</td>
			<td>CDC-9950-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">NAPCO series 8000WJ Incubator</td>
			<td style="width:199px;">Thermo scientific</td>
			<td style="width:207px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Allegra X-15R Centrifuge</td>
			<td style="width:199px;">Beckman Coulter</td>
			<td>392932</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Mini Mouse Bench centrifuge</td>
			<td style="width:199px;">Denville</td>
			<td style="width:207px;">C0801</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

identification of a murine erythroblast subpopulation  enriched in enucleating events by multi-spectral imaging flow cytometry

红细胞生成哺乳动物总结了眼球摘除的戏剧性过程，结果在网织红细胞的形成。眼球摘除的机制尚未完全阐明。学习去核红细胞显微镜中的关键蛋白和结构的本地化时遇到的一个常见问题是，观察细胞进行眼球摘除足够数量的难度。我们已经开发了一种新的协议分析使用流量多参数高速细胞成像（多光谱成像流式细胞仪），结合免疫荧光流式细胞仪中，为了确定有效的一个显著号码去核的事件的方法，它允许获得测量和进行统计分析。 首先，我们在这里描述，以便用于同步小鼠红细胞和增加捕获眼球摘除在日的概率两人在体外培养红细胞生成方法评估电子时间。然后，我们详细描述了为了眼球摘除由多光谱成像流式细胞仪过程中，研究细胞内蛋白质或脂质筏的本地化固定和透后红细胞的染色。随着尺寸和它们用来识别正色红细胞DNA/Ter119染色，我们利用在明视场通道，辅助在识别细长细胞和“三角形的质心的XY Ter119/Draq5的小区的参数”纵横比“ “允许的细胞事件，其中TER119染色（新生网织红细胞）的中心相距甚远DRAQ5染色（细胞核进行挤压）的中心，鉴定因而指示一个即将去核细胞。的正色红细胞种群具有高增量的质心和低纵横比的子集被高度富集在去核细胞。

首先，电池是基于他们的明场长宽比（他们的未成年子女与他们的长轴长度之比）和明场区（指示其规模大小）进行分析。具有比20一个明视场角值的情况下和高于200事件大多是碎片和细胞聚集体，分别与被排除在分析（ 图2A）。单细胞（门“R1”），然后分析了基于他们对梯度RMS参数，它表示图像的锐度值。门“R2”是创建一个包含细胞与梯度RMS值超过了50 个百分位，以选择...

Watch this Scientific Journal Video about Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry at JoVE.com

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

本协议描述了一种新的识别去核红细胞正色由多光谱成像流式细胞仪，以提供红细胞去核过程的可视化的群体的方法。

一个小鼠红细胞亚群富集去核活动由多光谱成像流式细胞仪鉴定

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

identification-murine-erythroblast-subpopulation-enriched-enucleating

Research

JoVE Journal

Biology

12.5K 次观看.  University of Cincinnati College of Medicine. 本协议描述了一种新的识别去核红细胞正色由多光谱成像流式细胞仪，以提供红细胞去核过程的可视化的群体的方法。 

视频: Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM：免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

科研

生物学

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.