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 <sup...

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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">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">&#945;MEM medium</td>
			<td style="width:199px;">CellGro&#160;</td>
			<td style="width:207px;">15-012-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:363px;">IMDM medium</td>
			<td style="width:199px;">Hyclone (Thermo Scientific)</td>
			<td style="width:207px;">SH30228.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Stempro-34 SFM</td>
			<td style="width:199px;">GIBCO (Life Tech)</td>
			<td style="width:207px;">10640</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:363px;">Stempro-34 nutrient supplement</td>
			<td style="width:199px;">GIBCO (Life Tech)</td>
			<td style="width:207px;">10641-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:199px;">Atlanta Biologicals</td>
			<td style="width:207px;">512450</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">BIT9500</td>
			<td style="width:199px;">Stemcell Technologies</td>
			<td style="width:207px;">09500</td>
			<td style="width:167px;"></td>
		</tr>
		<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>
		</tr>
		<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>
			<td style="width:199px;">Hyclone (Thermo Scientific)</td>
			<td style="width:207px;">SV30010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:363px;">L-glutamine</td>
			<td style="width:199px;">Hyclone (Thermo Scientific)</td>
			<td style="width:207px;">SH30590.01</td>
			<td style="width:167px;"></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:363px;">BD Pharmlyse (RBC lysis buffer)</td>
			<td style="width:199px;">BD Biosciences</td>
			<td style="width:207px;">555899</td>
			<td style="width:167px;"></td>
		</tr>
		<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>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:207px;">BP 531-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Hydrocortisone</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:207px;">H4001</td>
			<td style="width:167px;"></td>
		</tr>
		<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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Interleukin-3 (IL-3)</td>
			<td style="width:199px;">Peprotech</td>
			<td style="width:207px;">213-13</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:363px;">EPOGEN Epoetin Alfa (Erythropoietin, EPO)</td>
			<td style="width:199px;">AMGEN</td>
			<td style="width:207px;">available by pharmacy</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:363px;">CD44-FITC antibody</td>
			<td style="width:199px;">BD Pharmingen</td>
			<td style="width:207px;">553133</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:363px;">CD71-FITC antibody</td>
			<td style="width:199px;">BD Pharmingen</td>
			<td style="width:207px;">553266</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:363px;">Ter119-PECy7 antibody</td>
			<td style="width:199px;">BD Pharmingen</td>
			<td style="width:207px;">557853</td>
			<td style="width:167px;"></td>
		</tr>
		<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>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">&#946;-tubulin-AF488 antibody</td>
			<td style="width:199px;">Cell Signaling</td>
			<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>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">&#947;-tubulin antibody</td>
			<td style="width:199px;">Sigma</td>
			<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>
			<td style="width:199px;">Biostatus</td>
			<td style="width:207px;">DR50200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Ferrous sulfate</td>
			<td style="width:199px;">Sigma</td>
			<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>
			<td style="width:199px;">BD Falcon</td>
			<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>
			<td style="width:199px;">BD Falcon</td>
			<td style="width:207px;">352098</td>
			<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>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tuberculin syringe</td>
			<td style="width:199px;">BD</td>
			<td style="width:207px;">309602</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Insulin syringe</td>
			<td style="width:199px;">BD</td>
			<td style="width:207px;">329461</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Syringe needle 25G5/8</td>
			<td style="width:199px;">BD</td>
			<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 ...

Research

JoVE Journal

Biology

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

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

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

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

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

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

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

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

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

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

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

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...