这项工作得到了中国国家自然科学基金 （No. 81800207） 对 J.H. 的资助。非常感谢 Beckman Coulter， Inc. 在为流式细胞术和参数校准提供支持方面的帮助。我们感谢四川大学华西口腔医院口腔疾病国家重点实验室的 Jiao Chen 和四川大学华西医院再生医学研究中心的 Yu Qi，感谢他们在流式细胞术数据采集方面的帮助。

本研究中的实验总共使用了 10 只年龄在 8 至 12 周之间的 C57BL/6 小鼠。实验操作是根据四川大学机构动物护理和使用委员会 （#201609309） 批准的方案进行的。本文中使用的实验材料和流式细胞术参数列在 材料表中。
1. 小鼠骨髓细胞的分离和收集
<ol><li>严格按照机构指南对小鼠实施安乐死。为了诱导小鼠失去知觉，从 2% 异氟醚开始施用吸入麻醉剂，然后逐渐增加剂量至 5% 异氟醚，直到呼吸停止。</li>
	<li>解剖手术室或通风柜中的小鼠。用 70% 乙醇清洁和消毒小鼠。</li>
	<li>用锋利的剪刀完全剪断腿部的皮肤和肌肉，并解剖股骨。</li>
	<li>在研钵中用研磨棒轻轻压碎股骨，并用 DMEM 培养基冲洗骨髓细胞，直到骨头变白。</li>
	<li>用 100 μm 过滤器将获得的骨髓细胞过滤到 15 mL 试管中。在 4 °C 下以 800 x g 离心 5 分钟。</li>
	<li>弃去上清液，用 1 mL 红细胞裂解缓冲液在 4 °C 下裂解残留的红细胞 1 分钟。 在 4 °C 下以 800 x g 离心 5 分钟。 离心后，再次用 DMEM 培养基洗涤。</li>
	<li>将细胞重悬于 2 mL 孵育溶液（DMEM 培养基 + 5% FBS）中，用自动细胞计数器计数细胞，并使用孵育溶液将细胞浓度调节至 1.0 x 106 个细胞/mL。</li>
</ol>2. 细胞染色
<ol><li>用 5 μg/mL Hoechst 33342 补充每只小鼠的 2 mL 骨髓细胞悬液。向另一个 1 mL 等分试样中，加入 100 μM 维拉帕米作为阴性对照。</li>
	<li>在 37 °C 的水浴中孵育 90 分钟，每 30 分钟轻轻搅拌一次。</li>
	<li>孵育后，将细胞在冰上冷却 5 分钟，然后在 4 °C 下以 250 x g 离心 5 分钟。 将细胞重悬于冷运行溶液 （HBSS + 2% FBS） 中。在 4 °C 下离心 5 分钟并弃去上清液。</li>
	<li>将所得细胞沉淀重悬于每个样品 500 μL 的电泳溶液中。在 FCM 分析之前，用 2 μg/mL 碘化丙啶 （PI） 补充细胞，并在冰上放置约 5 分钟。</li>
</ol>3. 流式细胞术
注意：有关使用的各种系统和配置的摘要，请参见 表 1。
<ol><li>使用 FCM 中的每日质量控制 （QC） 荧光球校准所需通道的变异系数 （CV） 值，并在成功完成质量控制后进行样品测试。<ol><li>选择软件的桌面快捷方式并启动软件。</li>
		<li>在 QC/Standardization 菜单中选择 Start QC/Standardization 以访问 QC 实验。</li>
		<li>将 QC 荧光球样品管插入试管架中。</li>
		<li>选择 Start（开始 ）以加载样品并开始运行 QC 程序。FCM 在 QC 通过后即可使用。</li>
	</ol></li>
	<li>分别用 355 nm、375 nm 和 405 nm 激光激发相同样品的 Hoechst 33342 染料，以检测 690/50 nm 通道中的 Hoechst 红和 450/50 nm 通道中的 Hoechst 蓝。<ol><li>通过在 File 菜单中选择 New Experiment 来创建新实验，指定文件路径，然后保存实验。</li>
		<li>在 Settings 菜单中选择 Set Channel 。选中通道信号复选框（Y585、V450、V660、NUV450、NUV660、UV450、UV660），然后在标签列（Y585：PI;V450：Hoechst Blue-405 nm;V660：Hoechst 红 - 405 nm;NUV450：Hoechst Blue-375 nm;NUV660： Hoechst Blue-375 nm;UV450：Hoechst Blue-355nm;UV660：Hoechst Blue-355 nm）。</li>
		<li>点击绘图区域中的 伪彩色绘图 图标以创建绘图。选择轴名称以更改显示的渠道。</li>
		<li>单击 Test Tube 屏幕中的 Add Tube 以创建新的样品管并更改其名称。</li>
		<li>选择 Run 以加载样品、查看图并建立门。调整增益和阈值设置。选择 Record 以保存数据。</li>
	</ol></li>
	<li>设计门设置逻辑。<ol><li>对于第一个图，点击 X 轴 选择 FSC-W，然后点击 Y 轴 选择 FSC-A。选择 多边形门 以绘制门 A 以圈出单个细胞并排除粘附细胞（图 1A）。</li>
		<li>对于第二个图，点击 X 轴 选择 FSC-A，然后点击 Y 轴 选择 SSC-A。选择 Polygon Gate 以绘制门 B 以分离非碎片细胞并排除细胞碎片（图 1B）。</li>
		<li>对于第三个图，点击 X 轴 选择 PI-A，然后点击 Y 轴 选择 SSC-A。选择 多边形门 以绘制门 D。选择显示负 PI 的 活细胞 （图 1C）并绘制门 C 以获得活细胞。</li>
		<li>对于第四个二维图，点击 X 轴 选择 Hoechst Red，然后点击 Y 轴 选择 Hoechst Blue。右键单击 Plot ，然后从下拉菜单中选择 Property 。选择 Linear Format f或同时选择 X 轴和 Y 轴。选择 Polygon Gate 以绘制门 SP 以获得 SP 单元（图 1D）。</li>
	</ol></li>
	<li>为了评估样品的合格性，使用 355 nm 的特异性流式细胞仪23 进行 SP 细胞检测。</li>
	<li>同时使用 355 nm（激光功率：20 mW）和 405 nm（激光功率：80 mW）激光检测对照细胞（添加维拉帕米）和实验细胞。</li>
	<li>在对应于两个激光器的 690/50 nm 和 450/50 nm 通道处获取荧光信号。用透明 SP 细胞观察 355 nm 和 405 nm 激光对 Hoechst 33342 染料的有效刺激（图 2B-C）。</li>
	<li>对于相同的样品，使用 375 nm（激光功率：60 mW）和 405 nm（激光功率：80 mW）激光器进行细胞检测。</li>
</ol>

用于轻松检测侧群细胞的替代激光激发方法

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没有申报利益冲突。

我们使用所描述的方案进行了三个实验，每个试验涉及 3-4 只小鼠，总共产生 10 只小鼠。SP 细胞的比例范围为 0.05% 至 0.76%。值得注意的是，在小鼠中观察到个体差异。我们使用了四台流式细胞仪来分析 Hoechst 样品。据观察，在新型流式细胞术中，20 mW 355 nm 激光对 Hoechst 染料的激发与老式流式细胞术中 100 mW 355 nm 激光器的激发作用相当。这种现象可归因于两种系统中采用的激光器类型的差异。观?...

通过 Hoechst 33342 染色鉴定侧群 （SP） 细胞，并使用流式细胞术 （FCM） 进行分析。SP 细胞的特征是通过其 ATP 结合盒 （ABC） 转运蛋白将荧光 DNA 染料泵出细胞 1,2。该方法最初用于分离小鼠骨髓造血干细胞 （HSC）1。骨髓 SP 细胞富含以 CD117+Sca-1+Lin-Thy1 低表达 3,4 为特征的 HSC 群体。此后，该方法被广泛用于从其他组织中分离和富集干细胞，包括心肌5、肝脏6、肺7、肾脏8 和前脑9。特别是，该方法在过去十年中已被应用于分离癌症干细胞 （CSC）。CSCs 代表一小群细胞，具有肿瘤发生、自我更新、化疗耐药和转移潜力10。CSCs 最初是在造血系统恶性肿瘤11 中发现的，随后在各种实体瘤中观察到，例如前列腺癌 12、卵巢癌13、胃癌14、乳腺癌15 和肺癌16。尽管有多种技术可用于 CSC 鉴定，但 SP 技术仍然是一个受欢迎的选择，因为它在各种组织和细胞系中具有广泛的适用性。此外，它是一种使用荧光激活细胞分选 （FACS） 分离 CSC 的有价值的技术16,17,18。实验结果表明，SP 细胞表现出明显的致瘤性，并且干细胞相关基因的表达水平升高19,20。我们之前的研究21 还表明，多发性骨髓瘤中分离的 SP 细胞的转录组学分析揭示了与干细胞相关的信号通路的富集，例如 hedgehog 通路。同时，我们对急性髓性白血病 SP 细胞中差异表达的基因进行了通路富集分析22。改变的基因富集在干细胞相关途径 （Wnt/β-catenin 、 TGF-β 、 Hedgehog 、 Notch ）。我们发现 1 x 105 个 SP 细胞可以在 BALB/c 无效小鼠中形成肿瘤22，而非 SP 细胞则不能，表明 SP 细胞具有白血病干细胞的特征。Hoechst SP 方法鉴定 CSC 的有效性是显而易见的。
Hoechst SP 协议随着研究的进展而得到完善和增强。该方案需要严格控制染料浓度、细胞密度、孵育温度和持续时间、缓冲液组成和 pH 值。对根据该方案制备的细胞样品进行流式细胞术分析。由于使用 355 nm 紫外激光激发 Hoechst 染料，并使用 690/50 nm 滤光片（Hoechst 红）和 450/50 nm 滤光片（Hoechst 蓝）检测其荧光发射，因此 FCM 需要 350 nm 激光。然而，由于 350 nm 激光器的成本高昂，大多数 FCM 并不普遍配备 350 nm 激光器。因此，我们试图找到一种替代的染料激发方法，以通过流式细胞术有效检测 SP 细胞。在这项研究中，评估了 375 nm 和 405 nm 激光器检测 SP 细胞的能力，并与 355 nm 激光器进行了比较。我们的研究结果表明，355 nm、375 nm 和 405 nm 激光检测到的 SP 细胞之间具有显著的相似性。这些结果表明，高功率 375 nm 和 405 nm 激光器可以作为 355 nm 激光器的可行替代方案，用于 SP 细胞检测。Hoechst 33342 的额外激发光源有助于使用更多的流式细胞术模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic cell counter</td>
			<td>Countstar</td>
			<td>1M1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Filter(100 &#181;m)</td>
			<td>BIOFIL</td>
			<td>CSS-013-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Daily quality control fluorospheres</td>
			<td>Beckman Coulter</td>
			<td>B5230</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium with 4.5g/L glucose (DMEM medium)</td>
			<td>CORNING</td>
			<td>10-013-CVRC</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>CORNING</td>
			<td>35-081-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Hyclone</td>
			<td>SH30030.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td></td>
		</tr>
	</tbody>
</table>

effective detection of hoechst side population cells by flow cytometry

通过 Hoechst 33342 染色鉴定侧群 （SP） 细胞，并使用流式细胞术 （FCM） 进行分析。Hoechst SP 方法用于基于 ATP 结合盒 （ABC） 转运蛋白的染料外排特性分离干细胞。该方法最初用于鉴定和分离造血干细胞 （HSC），但现在它已发展为主要侧重于癌症干细胞 （CSC） 的鉴定和分离。FCM 的传统检测方法是使用 355 nm 激光激发染料以检测 SP 细胞。通过这项研究，我们成功确定了染料激发的替代方法，可以有效替代使用 355 nm 激光检测 SP 细胞。这是通过使用高功率 375 nm 或 405 nm 激光器实现的。这使我们能够在检测 SP 细胞时发挥更高的选择性，而不仅仅是局限于 355 nm 激光流式细胞术。

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The SP cells are characterized by the pumping of the fluorescent DNA dye out of the cells through their ATP-binding cassette (ABC) transporter1,2. The method was originally established for isolating murine bone marrow hematopoietic stem cells (HSCs)1. The bone marrow SP cells were enriched with a population of HSCs characterized by &#160;CD117+Sca-1+Lin-Thy1low&#160;expression3,4. Thereafter, the method was widely used to isolate and enrich stem cells from other tissues, including cardiac muscle5, liver6, lung7, kidney8, and forebrain9. In particular, the method has been applied to isolate cancer stem cells (CSCs) in the past decade. CSCs represent a small population of cells that possess the properties of tumor initiation, self-renewal, resistance to chemotherapy, and metastatic potential10. CSCs were initially identified in hematopoietic malignancies11 and subsequently observed in various solid tumors such as the prostate12, ovarian13, gastric14, breast15, and lung16 carcinomas. Despite the availability of various techniques for CSC identification, the SP technique remains a favored choice owing to its broad applicability across diverse tissues and cell lines. Moreover, it is a valuable technique that isolates CSCs using fluorescence-activated cell sorting (FACS)16,17,18. The experimental findings demonstrate that SP cells exhibit pronounced tumorigenicity and display elevated expression levels of stem cell-associated genes19,20. Our previous study21 has also demonstrated that transcriptomic analysis of isolated SP cells in multiple myeloma reveals enrichment of signaling pathways associated with stem cells, such as the hedgehog pathway. Meanwhile, we performed pathway enrichment analyses on the genes differentially expressed in the SP cells of acute myelogenous leukemia22. The altered genes were enriched in stem cell-related pathways (Wnt/&#946;-catenin, TGF-&#946;, Hedgehog, Notch). We found that 1 x 105 SP cells could form tumors in BALB/c null mice22, whereas non-SP cells could not, indicating that SP cells had characteristics of leukemia stem cells. The efficacy of the Hoechst SP method in the identification of CSCs is evident.
The Hoechst SP protocol has been refined and enhanced through the progression of research. The protocol entails stringent control of the dye concentration, cell density, incubation temperature and duration, buffer composition, and pH value. The cell samples prepared in accordance with this protocol were subjected to flow cytometric analysis. Due to the excitation of Hoechst dye with a UV laser at 355 nm and detection of its fluorescence emission using both a 690/50 nm filter (Hoechst Red) and 450/50 nm filter (Hoechst Blue), a 350 nm laser is required for FCM. However, 350 nm lasers are not commonly equipped in most FCMs because of their high cost. Hence, we attempted to find an alternate approach for&#160;dye excitation for the effective detection of SP cells by flow cytometry. In this study, the capability of the 375 nm and 405 nm lasers in detecting SP cells was assessed and compared with that of the 355 nm laser. Our findings demonstrate a remarkable similarity between the SP cells detected by the 355 nm, 375 nm, and 405 nm lasers. These results suggest that the high-power 375 nm and 405 nm lasers can serve as feasible alternatives to the 355 nm laser for SP cell detection. The inclusion of additional excitation light sources for Hoechst 33342 facilitates the use of more flow cytometry models.

作者聚焦：用于 Hoechst 染色分析侧群细胞的创新激光技术

通过流式细胞术有效检测 Hoechst 侧细胞群

在图 2 中，对照组用维拉帕米处理，维拉帕米阻断干细胞中的 ABC 转运蛋白以防止 Hoechst 33342 的消除。因此，非维拉帕米组中的干细胞排出 Hoechst 33342 并形成称为 SP 细胞的阴性细胞群。355 nm 激光有效地激发了 Hoechst 33342 染料，从而清晰地观察到骨髓的 SP 细胞（图 2A）。发现 355 nm 激光和 405 nm 激光检测到的相同样品的 SP 细胞基本相同（...

在这里，我们展示了高功率 375 nm 和 405 nm 激光器可以有效地激发 Hoechst 33342，并作为 355 nm 激光器的可行替代方案进行侧群 （SP） 细胞检测，从而扩大了流式细胞术应用中可用激光器的范围。

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized ...

effective-detection-of-hoechst-side-population-cells-by-flow-cytometry

Research

JoVE Journal

Cancer Research

Effective Detection of Hoechst Side Population Cells by Flow Cytometry

569 次观看.  Sichuan University. 我们使用 Hoechst 染色群体方法来检测肿瘤起始细胞。在本文中，我们试图寻找 Hoechst 激励的替代方法。我们发现高功率 375 纳米和 405 纳米激光器可以作为传统 355 毫米激光器的可行替代品。传统上，Hoechst 染料是用 355 纳米的紫外激光激发的。由于紫外激光器的成本高昂，大多数流式细胞术型号通常不配备紫外激光器。我们的方案扩展了可用于?...

视频: 作者聚焦：用于 Hoechst 染色分析侧群细胞的创新激光技术

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized for the isolation of stem cells based on the dye efflux properties of ATP-binding cassette (ABC) transporters. The method was initially employed for the identification and isolation of hematopoietic stem cells (HSCs), but it has now evolved to primarily focus on the identification and isolation of cancer stem cells (CSCs). The traditional detection method of FCM uses a 355 nm laser to excite the dye to detect SP cells. Through this study, we have successfully identified alternative approaches for dye excitation that can effectively replace the detection of SP cells using a 355 nm laser. This is achieved through the utilization of high-power 375 nm or 405 nm lasers. This allows us to exercise enhanced selectivity in the detection of SP cells rather than being solely limited to the 355 nm laser flow cytometry.

Here, we show that high-power 375 nm and 405 nm lasers can effectively excite Hoechst 33342 and serve as a viable alternative to the 355 nm laser for side population (SP) cell detection, thereby expanding the range of available lasers in flow cytometry applications.