作者要感谢列昂尼德·沃尔科夫博士和Eric布沙尔他们关于荧光显微镜技术咨询。 经济支持 由健康研究（批＃MOP-93781），以GB的加拿大学院资助

动物保健是按照舍布鲁克大学动物护理和使用委员会。 1.管子准备<ol><li>细胞分选前一天，加1-2热灭活的胎牛血清（FBS）中的溶液至5毫升聚丙烯圆底管中，15毫升和50毫升聚丙烯锥形管。 关键的一步：确保在协议中使用的每个管涂层。 注意：FBS涂层防止生殖细胞粘到管壁。 </li><li>慢慢地，管均匀使用旋转器以4°C到外套搭配O / N的反转。 </li><li>第二天，通过倾析从包被的管中删除FBS中。 <ol><li> *关键的一步：确保中的5毫升聚丙烯圆底管及用于细胞制备的15和50毫升管（协议步骤2.1至2.11）被完全清空FBS，以防止煽动（侧流也就是不精确的偏差）。使用P200的吸管去除FBS。 <br/&gt;注：残余之前排序可能导致细胞或在细胞分选使用的其它管的污染的损失FBS的管， </li><li>离开的FBS（约100-200微升）在5 ​​ml的聚丙烯轮用于收集纯化的细胞底管小的残留量。 </li></ol></li></ol> 2.细胞的制备<ol><li>用O 2 /异氟醚（归纳为5％，那么保持在2％），其次是二氧化碳窒息牺牲一只公鼠在麻醉下。 </li><li>消费和解封装双侧睾丸和讳言与在500μl分类缓冲器到1.5毫升管的小剪刀。 </li><li>冲洗生殖细胞从输精管通过温和上下吹打在1.5 ml管，先用截短100-1,000微升末端，然后用一个完整100-1,000微升小费。 </li><li>使用40微米的细胞过滤一个过滤步骤删除碎片和团块，收集滤液放入50ml锥形管上一个iously涂上FBS的第1步。 </li><li>与排序缓冲高达3毫升的总体积和传递滤液进入的15毫升锥形管先前在步骤1涂覆洗过滤一次。 </li><li>添加16微升50毫摩尔EDTA pH值为8每个细胞悬浮液的毫升（48微升3毫升），以便获得0.8 1mM EDTA的最终浓度。 </li><li>要修复的生殖细胞，用10ml的移液管轻轻搅动（涡流以低速），这将产生一个乳白色悬浮液慢慢加入3倍体积冰冷的100％乙醇。 关键的步骤：慢慢加入乙醇是至关重要的，重要的是保持细胞制剂的完整性。我们注意到，迅速加入乙醇导致造成分拣效率的大幅度下降细胞溶解。对于3 ml的细胞悬浮液，9毫升乙醇应在大约1分钟加入。 </li><li>孵育细胞在冰上15分钟，偶尔搅拌颠倒。 </li><li>在800 XG 5分钟离心细胞悬液，并取下透明上清。 </li><li>轻轻悬浮在2ml排序缓冲器的固定生殖细胞。 </li><li>加入4微升SYTO16 DNA染料（在DMSO 1mM的溶液）并孵育至少30分钟（避光）。 注意：最佳排序结果是使用SYTO16，因为它是一个可渗透DNA染料，很容易结合到精子细胞的高度压实的细胞核获得。 </li></ol> 3.细胞分选仪设置注意：在这里，一个4激光器（405纳米 - 紫色，488纳米 - 蓝色，561纳米 - 黄绿色，633纳米 - 红）20参数BDFACSAria III细胞分选仪被使用。在BD FACSDiva 6.1.3软件用于可视化和分析数据。的设置可以根据所用分拣机的类型而变化。 <ol><li>通过运行70％的乙醇作为流体关机过程之前排序（“流式细胞仪”软件中的菜单）在鞘液消毒的FACS分拣机。该软件的工具栏中选择“流式细胞仪”，然后选择“射流SHutdown“。按照由过程中提到的步骤。 </li><li>制备和筛选的1×PBS用0.22微米的过滤器，以便消除任何晶体和污染物。填充鞘罐在罐的内侧上部焊接线。 </li><li>启动软件并登录。启动FACS分拣机之前分拣预热激光器至少30分钟。运行两个流体启动过程来冲洗掉乙醇从流体。该软件的工具栏中选择“流式细胞仪”，然后选择“流控启动”。遵照过程中提到的步骤。 注：此射流恢复正常鞘液（1X PBS）。 </li><li>清洁排序块和偏转板，用蒸馏水彻底干燥。超声处理一个100微米的喷嘴在超声处理浴中放置在蒸馏水的试管2分钟。彻底擦拭喷嘴。 </li><li>插入100μm的喷嘴进入流动池和喷嘴锁定杆顺时针转动到12HR的地位。将外皮压到20 psi。 </li><li>打开流和调整幅度，以获得目标值的频率（约30），1滴（约150）和Gap（约12），并建立了稳定的液滴断离模式（几个卫星滴）。关闭衰减。 </li><li>确保流是直的，并通过改变所述排序块的角度击中废物抽吸器的中心。注：在10psi一个130微米的喷嘴进行了测试，并没有明显的差异。 </li><li>设置激光延迟为0的蓝色，-77.39为红，37.33为紫色和-39.85为黄绿色激光器。 </li><li>集区域扩展到1.14为蓝，1.0为红，0.75紫色和0.96的黄绿色激光器。 </li><li>使用测试排序（在软件的“侧流”窗口），请确保侧流不煽动。在“侧流”窗口，调整每边流的电压滑块，使他们击中了MIDDLË测试集管。如果中心流不紧，调整2 次 ，3 次和4 次下跌的设置来限制中心流。 </li><li>启动流式细胞仪设置和软件（“流式细胞仪”菜单）追踪（CS＆T）的应用程序。 </li><li>使用流式细胞仪的设置和跟踪珠粒在每350微升1滴自动化使用制造商的说明细胞仪性能的表征和跟踪。 </li><li>退出CS＆T的应用程序。应用CS＆T的设置，确保流参数仍允许一个稳定的液滴断离的模式，并打开甜蜜点按钮，在制造商的说明书中描述（在“断离窗”）。 </li><li>优化利用荧光珠的投递延迟值（见表材料 ）。 <ol><li>在保持器安装在收集管中。打开排序块门。在“侧流”窗口中，开启电压，然后选择“测试排序“，然后单击吸气抽屉按钮，打开抽屉，并有机会获得收集管。 </li><li>优化侧流，以便它们进入该管的中心。根据需要调整值和滑块。 </li><li>关闭排序块门，确保调整千分表以获得中央的亮珠点。取消选择废抽屉，测试排序和电压。取出收集管。 </li><li>根据制造商的指示优化下降延迟。 <ol><li>简单地说，撼动珠药瓶大力稀释1滴​​珠0.5毫升的PBS。 </li><li>使用制造商的说明，设置使用该软件提供的投递延迟实验模板下降延迟。另外，使用自动删除延迟功能设置下拉延迟。 </li><li>加载的荧光珠的管和调节流速直至该阈值速率是〜3000至4000次/秒。设置排序精度“初始”和最后立法院“微调”中的“排序布局”窗口。选择滤光器和排序珠。调整投递延迟，直到〜100％被分拣到左边。 注意：该步骤是关键的，以确保感兴趣排序细胞分化成的侧流。所述珠和滤光器被用于实现接近100％的微滴的偏差。 </li></ol></li></ol></li><li>放置FSC 1.5 ND（中性密度）滤光片在FSC检测器模块的左端。将窗口扩展到2.00微秒和FSC面积比例为1.00（在“激光”选项卡中的“流式细胞仪”窗口）。 </li><li>在装入样品管，放置50μm的样品过滤器线在样品线的末端，以避免结块。要做到这一点，选择在“流式细胞仪”菜单中的“更改样品过滤器”。 </li><li>测试该样品流体的组成，以实现良好的分离，而不侧流之间扇形并防止细胞粘到polypropyle东北管。 <ol><li>装入样品管，打开偏转板，然后选择“测试类”与抽屉关闭。如果发生分离没有太多的扇形看一边流。 注意：范宁可能是由于在样品缓冲液中的高蛋白质浓度。 </li></ol></li><li>装载样品管进入FACS仪器。选择300rpm的样品搅拌和4℃的样品温度。选择“获取仪表板”，“采集数据”。 </li><li>如果需要的话，稀释样品以达到一个适当的事件速率至最大5,000个事件/秒（以3.0的最大流量）。严格遵守这些限制，以维护选细胞的纯度。 </li><li>为了分析，将细胞进行排序，优化FSC阈值，以消除碎屑而不与所研究的总体干扰。在对数标度，调整光电倍增管（PMT）探测器与三十零分之五百三十○滤波器的电压，以便使肯定的总人口的荧光信号的比例内配合，和栅极上的阳性群体（图1，门1）。 </li><li>生产前向散射区域（FSC-A）/侧面分散区域的SYTO16积极的人口（SSC-A）的情节和调节FSC-A约110伏的线性度，和SSC-A约150V的在对数标度的可视化所有的事件，同时排除非常大的细胞和细胞聚集。 </li><li>在“排序布局”窗口中，将“排序精度模式”到“4路纯度”（产量面膜：0，纯度面膜：32，相面膜：0）。从“目标活动”菜单进行连续排序选择“连续”。 </li><li>添加种群在对应于管中的字段进行排序（在“排序布局”窗口）。地方人口在中间较高频率和那些与在端部较低的频率。设置电压板2500五，在整理，保持样品收集牛逼冰上ubes。 </li></ol> 4.细胞分选<ol><li>随即排序前，用一个事先涂5毫升聚丙烯50微米的过滤器圆底管过滤器单元。 </li><li> 1-2毫升分类缓冲器的广泛冲洗过滤器。 </li><li>使用488纳米激光装备细胞分选仪按照图1中详述的门控方案排序固定的生殖细胞。在收集事先涂在第1冰上5毫升聚丙烯圆底管纯化馏分。 <ol><li>确保100-200微升的FBS的被保持在收集管，以避免在分选的细胞在管壁的粘附。 </li><li>确保SYTO16的浓度是饱和的，以避免在分拣期间荧光的波动。 SYTO16是饱和的时，有细胞染色强度中的所述的Alexa Fluor 488参数（DNA染色）中的排序的图形没有进一步的变化。如有必要，添加1微升SYTO16至分拣管达到饱和。 </li><li>显示器上显示以下参数的图形总事件：事件VS的Alexa Fluor 488-A的数量。 </li><li>与门1选择阳性DNA染色细胞， 如图1。 <ol><li>显示单元从1号门上使用的SSC-A对FSC-A作为参数的图形<ol><li>与门2选择细胞，显示出在图1中。 </li><li>显示单元从2号门上使用FSC-A对的Alexa Fluor 488-A作为参数的图形。 </li><li>选择单元格与5号门和6号门上的门2的图形，如显示在图1中。 </li><li>另5号门和6号门上显示使用的Alexa Fluor 488-W VS的Alexa Fluor 488-A作为参数的图形。 </li><li>选择精子步骤1-9精子在5号门的图形和精子步骤10-12精子细胞在6号门图形，显示在图1中。 </li></ol></li><li>显示使用FSC-A对的Alexa Fluor 488-A作为参数，从1号门的细胞上的图形<ol><li>与门3和门4选择细胞，表明在图1中。 </li><li>另外3号门和4号门显示使用的Alexa Fluor 488-W VS的Alexa Fluor 488-A作为参数的图形。 </li><li>选择精子步骤13-14精子在3号门的图形和精子步骤15-16精子细胞在4号门的图形，如显示在图1中。 </li></ol></li></ol></li></ol></li></ol>

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The authors declare no competing financial interests.

生精细胞始终是具有挑战性的研究给出的生精上皮的复杂性， 以及在体外培养的有限的成功。多年来，许多方法来净化来自不同物种生殖细胞被开发。利用重力纯化用的Percoll或牛血清白蛋白梯度沉降技术通常提供完整生发细胞的良好的产率，但缺乏某些类型的细胞之间的适当定义如减数分裂四倍体细胞和精子细胞10。而且，这些技术需要特殊的装置（通常自制的）可能不容?...

Haploid round spermatids differentiate into spermatozoa by a process called spermiogenesis. This involves many different steps including the acquisition of a flagellum, chromatin and cytoskeleton remodeling, condensation of the nucleus as well as the loss of most of the cytoplasm. These unique cellular events must be finely regulated in order to produce a mature functional gamete with an intact genome suitable for fertilization. Spermiogenesis can hardly be studied in vitro since no reliable cell culture system has so far been able to support progression through the different steps of the process. Moreover, actual in vitro techniques lead to a poor yield1,2. In vivo, proper transitions through the different steps of spermiogenesis are crucial for the natural functional integrity of the male gamete. Successful purification of spermatids according to their differentiation steps has never been accomplished with a level of enrichment sufficient to allow molecular characterization of spermiogenesis. For instance, purification of key steps of the spermatidal differentiation would be especially useful to study the developing acrosome, formation of the midpiece3, cell junction dynamics4, RNA dynamics5, chromatin remodeling process6,7 or genomic stability8. Purification of spermatids has been hampered by their progressive morphological transformation, the lack of known stage-specific external biomarkers, and their peculiar shape and size. 
Although most male germ cells display a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids. This stems from our early observation that seminiferous tubule sections show variable intensity of DNA staining throughout the different spermiogenesis steps. Although DNA staining is consistent with their haploid set of chromosomes from spermiogenesis steps 1 to 7 (round spermatids), we observed a sharp increase in fluorescence intensity with DAPI or SYTO 16 around the onset of nuclear reorganization and chromatin remodeling (spermiogenesis step 8) reaching a peak at the onset of nuclear condensation (spermiogenesis steps 11-12). Following condensation of the nucleus, DNA staining intensity decreases until spermiation (spermiogenesis step 16). We surmised that this was likely associated with the formation of their peculiar chromatin structure transition where histones are replaced by protamines. We therefore developed a reliable flow cytometry method that allows the separation of spermatids using the variation of DNA intensity of spermatids as a main selection parameter.
A simple flow cytometry approach is described to separate mouse spermatids with high purity (95-100%) based on their apparent DNA content (SYTO16 staining), size and granulosity. Spermatids are separated into four populations; spermiogenesis steps 1-9, 10-12, 13-14 and 15-16. Purified spermatids are suitable for genetic/genomic analysis, as well as proteomic applications as described in a recent publication from our group9.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Isoflurane</td>
			<td style="width:119px;">ABBOT</td>
			<td style="width:129px;">05260-05</td>
			<td style="width:184px;">For mouse anesthesia before euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Fetal bovine serum</td>
			<td style="width:119px;">Wisent</td>
			<td style="width:129px;">90150</td>
			<td style="width:184px;">For tube coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">1X PBS</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">EDTA</td>
			<td style="width:119px;">BioShop</td>
			<td style="width:129px;">EDT</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">HEPES</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:129px;">H</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">100 % Ethanol</td>
			<td style="width:119px;">Les alcools de commerce</td>
			<td style="width:129px;">092-09-11N</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">SYTO 16</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:129px;">S7578</td>
			<td style="width:184px;">DNA staining</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">5 ml polypropylene round bottom tubes</td>
			<td style="width:119px;">BD Falcon</td>
			<td style="width:129px;">352063</td>
			<td style="width:184px;">Sorted cells collection</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">15 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">1500</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">50 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">5000</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">TEC4 anaesthetic vaporizer</td>
			<td style="width:119px;">Ohmeda</td>
			<td style="width:129px;">1160526</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">CO2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">C799117902</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">O2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">O254130501</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Homemade mouse gas chamber</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">40 &#181;m Falcon cell strainer</td>
			<td style="width:119px;">Corning Incorporated</td>
			<td style="width:129px;">352340</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">50-micron sample line filters</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">649049</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Vortex mixer</td>
			<td style="width:119px;">Labnet international, inc.</td>
			<td style="width:129px;">S0200</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dynac centrifuge</td>
			<td style="width:119px;">Clay Adams</td>
			<td style="width:129px;">101</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Celltrics 50 &#181;m filters</td>
			<td style="width:119px;">Partec</td>
			<td style="width:129px;">04-004-2327</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">488 nm laser-euipped cell sorter</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">FACSAria III</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:135px;">Accudop Fluorescent Beads</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">345249</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">Sorting Buffer: 1X PBS, 1mM EDTA pH 8.0, 25mM HEPES pH 7.0, 1%FBS</td>
			<td></td>
			<td></td>
			<td style="width:184px;">FBS is heat-inactivated. Make fresh solution, 0.22 &#956;m filtered and keep at 4&#176;C.</td>
		</tr>
	</tbody>
</table>

step-specific sorting of mouse spermatids by flow cytometry

小鼠精子细胞分化是用于生产的官能雄性配子具有完整基因组中的一个关键的工艺要发送给下一代。到目前为止，这种形态转变的分子研究受到阻碍，缺乏一种方法允许精子细胞分化为后续分析这些重要步骤，充分分离。因为在精子细胞进行染色质重塑一个奇特的增加，DNA荧光在这些细胞使用流式细胞仪的正常浇注较早尝试可能是困难的。基于这一观察，我们提供了一个简单的流式细胞术方案细节，让四个种群的小鼠精子细胞固定用乙醇可重放纯化，每个代表在核重塑过程不同的状态。富集的人口使用步骤的具体指标和形态准则确认。纯化的精子细胞，可用于基因组和蛋白质组IC分析。

使用流式细胞仪门控策略 图1表示了流式细胞用于四个高纯度精子细胞种群进行排序选通策略。简而言之，将细胞与阳性DNA染色（的Alexa Fluor 488-A）的第一个带门从精子1.精子细胞的步骤1-12被选择（栅极2）上选择一个点图表示granulosity（SSC-A）与大小（FSC -A）从门1。然后，从精子的精子细胞的步骤1-9和精子步骤10-12?...

Watch this Scientific Journal Video about Step-specific Sorting of Mouse Spermatids by Flow Cytometry at JoVE.com

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

步特定老鼠的精子细胞分选流式细胞仪

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

step-specific-sorting-of-mouse-spermatids-by-flow-cytometry

Research

JoVE Journal

Developmental Biology

10.4K 次观看.  Université de Sherbrooke. We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

视频: Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

科研

发育生物学

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

隔离并通过流式细胞仪分析胰腺间充质细胞

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

使用标准实验室设备从小鼠测试中浓缩帕西滕苯细胞和精子

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

使用流动细胞测量来检测和量化正在发育的斑马鱼中改变的血液形成

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

从未受干扰和受伤的小鼠骨骼肌中分离和培养纤维脂肪祖细胞和肌肉干细胞的FACS-分离和培养

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

流式细胞术从小鼠组织中鉴定和分离爆破形成单元和集落形成单元红系祖细胞

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...