Name: isolation and staining of mouse skin keratinocytes for cell cycle specific analysis of cellular protein expression by mass cytometry
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Description: Watch this Scientific Journal Video about Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry at JoVE.com

这项工作的支持来自皮肤病系、科罗拉多大学盖茨再生医学中心和科罗拉多大学皮肤病中心形态和表型核心(NIAMS P30 AR057212)。作者感谢 UC 癌症中心流式细胞测量共享资源和支持赠款 (NCI P30 CA046934) 用于大规模细胞仪的运行,并感谢核心的 Karen Helm 和 Christine Childs 在流量和质量细胞测量方面的专家建议技术。

科罗拉多大学安舒茨医学院机构动物护理和使用委员会批准了本协议中描述的动物实验。
1. 准备工作
<ol><li>设计一个金属标记的抗体面板。使用免费的在线面板设计软件12,包括127碘氧尿素 (IdU)、164 Dy(Dysprosium) 标记的抗 CCNB1 (CYCLIN B1)、175 Lu(卢泰姆)磷(p)-HISTONEH3Ser28 (pHH3) 和150 Nd (钛)-pRETINOBLASTOMA蛋白Ser807/811 (pR...

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本文概述的协议可在8小时左右完成。最终结果是悬浮在KCs中富集的细胞,这些细胞可以以细胞周期依赖的方式分析蛋白质表达。以前的几项研究已经概述了从人类和小鼠皮肤分离的IC的方法16,25。这些研究还包括用于流式细胞测定26的SC分离方案。然而,以前没有详细描述过将IC分离与使用质量细胞测定分析蛋白质表达和细胞周期动力学相?...

在分析癌症等高塑性疾病的动物模型时,基因表达与细胞周期阶段的相关性仍然是一个挑战。这一挑战的一部分是共同检测具有增殖标记的感兴趣的蛋白质(POI)。增殖细胞可在各种细胞周期阶段找到,包括G1、S、G2和M.Ki67是最常用的增殖标记之一,在细胞周期的所有阶段表达。它已被广泛用于分析人类和小鼠组织1,2,3。然而,与其他一般增殖标记一样,Ki67不识别单个细胞周期阶段。更精确的方法将胸腺素核苷类类似物(如溴氧尿酸(BrdU)结合到正在积极复制其基因组的细胞中(即S相)4、5。使用核苷酸类似物的一个缺点是需要在分析前几小时管理它们以活的动物。使用抗体在固定组织部分通常检测到 Ki67 和 BrdU。这种方法的一个优点是,POI的位置可以在组织架构(例如,皮肤表皮的基础层)中确定。这种方法也不需要组织分离,可能导致基因表达的变化。一个缺点是组织固定或组织处理OCT冷冻或石蜡切片可能遮挡抗体靶点(即抗原)。抗原的回收通常需要热或组织消化。染色强度的量化也可能具有挑战性。这是由于染色、截面厚度、信号检测和实验偏置的变化。此外,在大多数典型的实验室设置中,可以同时检测到数量有限的标记。然而,较新的多路复用染色方法有望克服这些限制;例如成像质量细胞测定和提拉姆德信号放大6,7。
流式细胞测定是另一种检测增殖细胞的强大技术。它允许对同一细胞中的标记进行多倍检测,但大多数非造血细胞类型需要组织分离。增殖细胞的分析通常通过使用结合DNA的染料(例如,碘化钠(PI))8。流式细胞学还允许更精确地测定细胞周期阶段,当与BrdU结合9的检测相结合时。BrdU/PI 流式细胞测量虽然是一种强大的方法,但确实存在其缺点。如果不纳入相位特异性抗体,它无法解决G2/M和G0/G1相。然而,可以使用的抗体数量受到细胞自荧光、荧光辐射的光谱溢出和使用补偿控制的限制。这种限制标志着与POIs共同检测细胞周期标记的表达更具挑战性和艰巨性。一个较简单的方法是使用质量细胞测定10,11。该技术使用具有较窄检测光谱的金属结合抗体。一旦细胞被金属标记的抗体染色,它们就会蒸发,并且细胞测量时间(CyTOF)质谱仪检测到的金属。由于这些特性,质量细胞仪能够使用现有平台10、11对>40个不同标记进行多路检测。此外,还可以用金属对样品进行条形码,从而节省宝贵的抗体,同时减少样品到样品的染色变异性。另一方面,质量细胞学确实有一些缺点。对于非血液衍生细胞,市售的金属标记抗体数量有限。与使用荧光DNA染料相比,DNA含量的定量不太敏感,与荧光流细胞测定相比,质量细胞测量具有较低的信号检测动态范围。
此处描述的方案旨在分析从小鼠皮肤中新分离的角蛋白细胞 (KCs) 中的细胞周期动力学,并使用质量细胞测定来描述这些细胞中的细胞周期特定蛋白表达。该协议也可以与培养的细胞一起使用,或适应其他细胞类型。

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	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">12-well plate</td>
			<td style="width:237px;">Cell Treat</td>
			<td style="width:259px;">229512</td>
			<td style="width:368px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Intercalator solution</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201192A</td>
			<td>125 &#181;M - Ir intercalator solution</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Paraformaldehyde</td>
			<td style="width:237px;">Electron Microscopy Sciences</td>
			<td style="width:259px;">30525-89-4</td>
			<td>16 %&#160; PFA</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Strainer cap flow tubes</td>
			<td style="width:237px;">Fisher/Corning</td>
			<td style="width:259px;">352235</td>
			<td>35 &#181;m pore size&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cell sieve</td>
			<td style="width:237px;">Fisher</td>
			<td style="width:259px;">22363547</td>
			<td>40 &#956;m pore size&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cell detatchment solution</td>
			<td style="width:237px;">CELLnTEC</td>
			<td style="width:259px;">CnT-ACCUTASE-100</td>
			<td>Accutase</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Iodine Solution</td>
			<td style="width:237px;">ThermoFisher/Purdue</td>
			<td style="width:259px;">67618-151-17</td>
			<td>Betadine 7.5%-iodine surgical scrub</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Barcode permeabilization buffer&#160;</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201060</td>
			<td style="width:368px;">Cell-ID 20-Plex Pd Barcoding Kit</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Barcodes</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201060</td>
			<td style="width:368px;">Cell-ID 20-Plex Pd Barcoding Kit</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">pH strips</td>
			<td style="width:237px;">EMD</td>
			<td style="width:259px;">9590</td>
			<td>colorpHast</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">DMEM</td>
			<td style="width:237px;">Hyclone</td>
			<td style="width:259px;">SH30022.01</td>
			<td style="width:368px;">Dulbecco&#8217;s Modified Eagle Media</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Fine forceps</td>
			<td style="width:237px;">Dumont &#38; Fils</td>
			<td style="width:259px;">0109-5-PO</td>
			<td style="width:368px;">Dumostar #5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Curved precision forceps</td>
			<td style="width:237px;">Dumont &#38; Fils</td>
			<td style="width:259px;">0109-7-PO</td>
			<td style="width:368px;">Dumostar #7</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Calibration Beads</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201078</td>
			<td style="width:368px;">EQ Four Element Calibration Beads</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">HBSS</td>
			<td style="width:237px;">Gibco</td>
			<td style="width:259px;">14175-095</td>
			<td>Hank&#39;s Balanced Salt Solution</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Water</td>
			<td style="width:237px;">Fisher</td>
			<td style="width:259px;">SH30538.03</td>
			<td style="width:368px;">Hyclone Molecular Biology grade water</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Iododeoxyuridine</td>
			<td style="width:237px;">&#160;Sigma</td>
			<td style="width:259px;">I7125</td>
			<td>IdU</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cryo vials</td>
			<td style="width:237px;">ThermoFisher</td>
			<td style="width:259px;">366656PK</td>
			<td>internal thread</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cell Staining buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201068</td>
			<td style="width:368px;">Maxpar Cell Staining buffer</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Fix &#38; Perm buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201067</td>
			<td style="width:368px;">Maxpar Fix &#38; Perm buffer</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Fix I buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201065</td>
			<td style="width:368px;">Maxpar Fix I buffer</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Phosphate buffered saline</td>
			<td style="width:237px;">Rockland</td>
			<td style="width:259px;">MB-008</td>
			<td>Metal free 10x PBS</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">isopropanol-freezing container</td>
			<td style="width:237px;">ThermoFisher</td>
			<td style="width:259px;">5100-0001</td>
			<td>Mr.Frosty</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Sodium hydroxide</td>
			<td style="width:237px;">Fisher</td>
			<td style="width:259px;">BP359-500</td>
			<td>NaOH</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Petri dish</td>
			<td style="width:237px;">Kord-Valmark</td>
			<td style="width:259px;">2900</td>
			<td>Supplied by Genesee 32-107&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">15 mL conical</td>
			<td style="width:237px;">Olympus/Genesee</td>
			<td style="width:259px;">28-101</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">50 mL conical</td>
			<td style="width:237px;">Olympus/Genesee</td>
			<td style="width:259px;">28-106</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">6-well plate</td>
			<td style="width:237px;">Cell Treat</td>
			<td style="width:259px;">229506</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Cisplatin</td>
			<td style="width:237px;">Sigma</td>
			<td style="width:259px;">479306</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Dispase II</td>
			<td style="width:237px;">Sigma/Roche</td>
			<td style="width:259px;">4942078001</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">DMSO</td>
			<td style="width:237px;">Sigma</td>
			<td style="width:259px;">D2650</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">FBS</td>
			<td style="width:237px;">Atlanta Biologicals</td>
			<td style="width:259px;">S11150</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Hydrochloric acid</td>
			<td style="width:237px;">Fisher</td>
			<td style="width:259px;">A144-212</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Nuclear Antigen Staining&#160; permeabilization buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201063</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Nuclear Antigen Staining buffer</td>
			<td style="width:237px;">Fluidigm</td>
			<td style="width:259px;">201063</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:293px;">Trypan blue</td>
			<td style="width:237px;">Sigma</td>
			<td style="width:259px;">T8154</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Tuberculin syringe</td>
			<td style="width:237px;">BD</td>
			<td style="width:259px;">309626</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">Type IV Collagenase&#160;</td>
			<td style="width:237px;">Worthington Bioscience</td>
			<td style="width:259px;">CLSS-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and staining of mouse skin keratinocytes for cell cycle specific analysis of cellular protein expression by mass cytometry

该协议的目标是使用从小鼠皮肤表皮分离的单细胞,以细胞周期相关的方式检测和量化蛋白质表达变化。有七个重要步骤:表皮从真皮分离,表皮消化,用顺铂染色表皮细胞群,样品条形码,用金属标记的抗体染色细胞周期标记和蛋白质兴趣,通过质量细胞学检测金属标记抗体,以及分析不同细胞周期阶段的表达。与组织学方法不同,这种方法的优点是有可能在细胞周期的不同阶段对单个细胞中>40个不同标记的表达模式进行测定。这种方法还允许对蛋白质表达进行多变量相关性分析,这种分析比组织学/成像方法更具可量化性。该协议的缺点是需要暂停单个细胞,从而导致组织部分染色所提供的位置信息丢失。这种方法可能还需要加入额外的标记,以识别粗细胞悬浮液中的不同细胞类型。在超塑性皮肤病模型分析中,该方案的应用十分明显。此外,通过添加系骨特异性抗体,可调整该方案,用于分析特定亚型细胞(如干细胞)。该协议也可以用于分析其他实验物种的皮肤细胞。

表1显示了成人小鼠耳朵(图1)和非病理条件下新生儿皮肤的预期细胞产量和生存能力。该表还显示了来自混合 C57/126 背景的动物的代表性数据。预计其他菌株的皮肤会产生类似的细胞产量和活力。近似产量取决于皮肤的表面积,表明新生儿皮肤对于需要大量细胞的实验来说将是一个更好的选择(表1)。低产量或降低生存能力(<50%)?...

Watch this Scientific Journal Video about Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry at JoVE.com

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

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

该协议描述了如何从小鼠模型中分离皮肤角蛋白细胞,用金属标记的抗体染色,以及通过质量细胞测定法分析染色细胞,以便分析不同细胞周期阶段感兴趣的蛋白质的表达模式。

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 ...

此协议非常重要，原因有二。第一，它允许用户能够确定从动物模型中分离的皮肤细胞的细胞周期状态。它还使我们能够分析细胞周期离散阶段的蛋白质表达。与以往确定细胞增殖的方法相比，我们的方法允许更精确地测定细胞周期的不同阶段，还可以分析细胞分裂不同阶段的40多个不同标记。这大大提高了该方法的应用和多功能性。该协议可用于癌症研究和任何其他类型的疾病，其中需要确定细胞周期特定的蛋白质表达模式。此外，该协议可以修改为其他细胞类型，并在其他模型生物体。第一次用户应该专注于尽可能获得最高质量的细胞准备。这要求使用新鲜试剂及时处理实验皮肤，最少的组织消化时间，并在离心后仔细处置上流液，以保持细胞的完整性和数量。首先，使用免费的在线面板设计软件设计金属标记抗体面板，如手稿中所述。然后，在 60 摄氏度的正常氢氧化钠中，以每毫升 10 毫克的速度溶解 IdU 粉，然后准备 IdU 库存溶液。将IdU库存溶液等到微离心管后，在零下20摄氏度处冷冻，进行长期储存。使用前，立即将IdU溶液的pH值调整为7.5，用12个普通盐酸，并在丢弃的分条上测试，以确保溶液的pH值为7.5。要准备 2 x 对甲醛固定溶液，请将 5 毫升 10 x PBS 和 1 毫升 16%PFA 与 44 毫升纯分子级蒸馏水混合。要准备 100 毫摩尔顺铂库存溶液，在 10 毫升 DMSO 中溶解 300.5 毫克顺铂粉。对于在一天内使用的实验，准备一个10毫摩尔顺铂工作溶液。称重小鼠，然后确定每克体重0.1毫克IdU的剂量。通过内皮内注射管理IDU的计算剂量，并等待两个小时，然后收获细胞。使用一双手术级剪刀手术切除以前在耳底注射了IdU的安乐死小鼠的耳朵。将耳朵放在干燥的100毫米培养皿上。小心地将前部与后皮分开，使用细钳在切割区域的中间或边缘创建一个口袋，然后将两个皮肤皮瓣分开，然后继续处理前皮和后皮。小心地将一只耳朵的前部和后部皮肤放在 12 井培养板的一个井中，用一毫升新鲜准备好的脱皮/胶原酶溶液填充，皮肤的脱毛侧接触溶液。在37摄氏度下孵育一小时，消化皮肤。将消化的耳朵或新生儿皮肤放在干净的培养皿中，表皮侧接触盘子的表面。使用钳子，将皮肤压平，然后以圆形图案从中心工作到边缘，轻轻地将真皮从表皮上滑下来。用钳子，抓住边缘的表皮，轻轻将其从培养皿表面剥下来。小心地将表皮放在预加热的细胞分离溶液上，放在一个良好的板中。在37摄氏度下孵育5分钟，或在室温下孵育20分钟。使用无菌钳子抓住表皮，通过将表皮拖到盘子底部来分离细胞，进行擦洗。加入含有1%FBS的一毫升DMEM，通过40微米细胞筛入收集管。用额外的两毫升DMEM冲洗好，并加入细胞悬浮液。在120次g下离心5分钟后，小心吸上经。将细胞颗粒重新在含有1%FBS的一到两毫升DMEM中，并像以前一样再次对细胞进行颗粒化。要标记细胞以确定活细胞和死细胞，在含有25微摩尔顺铂和孵育一分钟的DMEM中重新发送100万到300万个细胞。通过以相等的 FBS 体积进行移液进行淬火。在120次g下离心5分钟后，将上经剂倒入含有稀释漂白剂的烧杯中，然后反转管子，将剩余的溶液排入纸巾。将细胞颗粒重新浓缩在两毫升无阳离子PBS中，然后像前面所述再次离心。要修复细胞，将细胞颗粒重新在一毫升无阳离子PBS中。在连续低功耗下旋转细胞，并添加两个 x PFA 固定缓冲液的一毫升。放在摇杆上，在室温下孵育10分钟。在500次g下离心5分钟后，小心地将上经剂去掉。用两毫升无阳离子PBS清洗细胞，在相同条件下再次离心，然后重复洗涤步骤。最后，将颗粒重新在两毫升无阳离子PBS中重新释放。将细胞以500倍g离心5分钟，并小心地将上一液去下。将细胞重新在一毫升的一个x固定缓冲液中，在室温下孵育10分钟，然后重复离心。要清洗细胞，请添加一毫升条形码渗透缓冲液。在500次g的细胞离心过程中，5分钟，通过添加100微升的条形码渗透缓冲器来准备条形码，并立即混合。将细胞颗粒重新填充在 800 微升的条形码渗透缓冲液中。在室温下向细胞添加条形码溶液，混合并孵育30分钟。在500次g下离心5分钟。在两毫升的细胞染色缓冲液中清洗细胞，并再次离心。接下来，在一毫升核抗原染色缓冲液中重新填充100万至300万个细胞，并在室温下孵育30分钟。在500次g下离心5分钟后，小心地将上经剂去掉。将细胞重新浓缩在一毫升核抗原染色渗透缓冲液中，并再次离心。重复再增和离心后，用温和的涡流将细胞颗粒重新在残余体积中。加入50微升细胞内抗体鸡尾酒，在室温下混合并孵育45分钟。添加两毫升细胞染色缓冲液。在与上一步相同的条件下再次离心，并取出上一代。将颗粒重新浓缩在两毫升细胞染色缓冲液中，以 500 倍 g 离心五分钟，然后重复重新增减和离心。将细胞重新在一毫升的间解中，在四摄氏度下储存一到三天。再次离心细胞后，用两毫升细胞染色缓冲液清洗颗粒，在相同条件下离心，用两毫升水进行两次额外的洗涤，同时进行同样的离心。用稀释的EQ珠溶液以每毫升一百万个细胞的浓度重新暂停细胞颗粒，然后在质量细胞仪上运行样品。成人小鼠耳朵和新生儿皮肤的预期细胞产量和生存能力被确定。近似产量取决于皮肤的表面面积。新生儿皮肤是需要更多细胞的实验的更好选择。在条形码质量细胞学数据规范化和去卷积后，为完整、单细胞和可行细胞选择基本浇注策略。活细胞的百分比可能表明染色前细胞的质量或状况。培养的癌细胞与分离的小鼠耳细胞之间的生存能力也显示出差异。活细胞在事件长度与CD45上被封闭，以排除造血细胞。包含血统标记允许在不同细胞周期阶段选择感兴趣的细胞群。在荧光流式细胞仪中，通过检测BrdU与PI获得基本细胞周期特征。然而，包含具有质量细胞学的其他增殖标记，可以更精确地识别细胞周期相。按照这种方法，可以为不同的细胞类型或实验条件构建细胞周期配置文件，例如同一实验中 CD45 阴性细胞与 CD45 阳性细胞的轮廓。在另一个示例中，对定义 G-One 相中 EGFR 和 mTOR PI3K 信号通路标记的分析揭示了 CD45 负细胞中类似的表达配置文件，以橙色和 CD45 正细胞显示，以蓝色显示。质量细胞学需要大量的高质量细胞。如果用户对稀有细胞组感兴趣，可能需要更多的细胞。分离的活细胞可用于DNA或RNA分析、生化研究，或可用于组织培养之前固定和染色的大规模细胞学。使用甲醛、盐酸或氢氧化钠时，用户应使用个人防护设备和安全柜（如有必要）。

Research

JoVE Journal

Developmental Biology

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

科研

发育生物学

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

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 ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

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

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 ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...