Name: tracking t cell division with cfse and flow cytometry
Uploaded: 2023-04-30T07:08:49.000Z
Duration: 10 min 57 s
Channel: JoVE

1. 准备
<ol>
	<li>开始实验前，戴上实验室手套并穿上适当的防护服。</li>
	<li>用去污剂对所有解剖工具进行初步消毒，然后用70%乙醇处理，最后彻底擦干。</li>
	<li>配制含有2%胎牛血清（FCS）的50 mL Hank's平衡盐溶液（HBSS）。</li>
</ol>
2. 解剖
<ol>
	<li>使用二氧化碳输送系统，通过缺氧法处死小鼠。将已处死的小鼠仰卧位固定在解剖板上，用剪刀和镊子进行纵向腹腔切开。</li>
	<li>用镊子将肠道和胃向腹部右侧移动，以暴露胃和脾脏。脾脏与胃相连。</li>
	<li>用镊子小心地将脾脏从胃部分离，并将其放入含有 5 mL HBSS 2% FCS 的培养皿中。</li>
</ol>
3. 免疫细胞分离
<ol>
	<li>将脾脏置于同一培养皿上的40 µm细胞筛网上，用研磨杵碾压脾脏以使其解离。</li>
	<li>将解离后的脾脏组织和液体转移至15 mL离心管中。</li>
	<li>在10°C条件下以370 × g 离心7分钟，弃去上清液，注意保留沉淀。</li>
	<li>用2 mL醋酸钾重悬沉淀以裂解红细胞。静置2分钟后，加入HBSS 2% FCS至总体积达15 mL。</li>
	<li>再次在10°C条件下以370 × g 离心7分钟，弃去上清液，将沉淀重悬于5 mL HBSS 2% FCS中。</li>
	<li>使用台盼蓝染色法计数细胞，并用适量体积的HBSS 2% FCS调整细胞终浓度至107 个细胞/mL。</li>
</ol>
4. CFSE 标记与T细胞刺激
<ol>
	<li>将分离的脾细胞以每管107个的密度分装至4个15 mL离心管中（标记为1至4号管）</li>
	<li>向每管中加入3 mL含2% FCS的HBSS。</li>
	<li>向每管中加入1 µL的CFSE（终浓度为5 µM）。</li>
	<li>将各管置于37°C、5% CO2培养箱中孵育10分钟。</li>
	<li>在3号和4号管中加入12 mL含2% FCS的HBSS，并加入抗CD3抗体，使其终浓度为2.5 µg/mL。3号和4号管通过抗CD3抗体进行刺激，以观察其对细胞周期的影响。</li>
	<li>向1号和2号管中加入12 mL含2% FCS的HBSS。1号和2号管中的细胞不进行刺激处理。</li>
	<li>将所有离心管在10°C下以370 × g离心7分钟，弃去上清液。</li>
	<li>用2 mL含2% FCS的HBSS重悬各管中的细胞沉淀。</li>
	<li>将所得悬液分别转移至6孔板中的不同孔内。</li>
	<li>将细胞在37°C、5% CO2条件下孵育3天。</li>
</ol>
5. 细胞染色
<ol>
	<li>第3天，在1号和3号孔中各加入2 mL HBSS 2%FCS。</li>
	<li>用力吹打并转移样品至5 mL流式管中。</li>
	<li>将2-4号孔中剩余的细胞继续在37°C、5% CO2条件下培养。这些细胞将在第5天进行分析，以研究刺激对细胞周期的长期影响。</li>
	<li>在10°C下以370 × g离心7分钟，弃去上清液。</li>
	<li>向每根管中加入100 µL抗体混合液（见表1）。</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>抗体</td>
			<td>荧光染料</td>
			<td>稀释比例</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Pacific Blue</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>BV786</td>
			<td>1/1600</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>PE</td>
			<td>1/400</td>
		</tr>
		<tr>
			<td>Thy1.2</td>
			<td>BV605</td>
			<td>1/400</td>
		</tr>
	</tbody>
</table>
表1： 抗体混合物组成。使用浓缩的抗体-荧光偶联物和HBSS配制的四种抗体鸡尾酒溶液。
<ol start="6">
	<li>将试管置于冰上避光孵育 20 分钟。</li>
	<li>加入 1 mL HBSS 2%FCS，于 10°C 下以 370 × g 离心 7 分钟。</li>
	<li>弃去上清液，将沉淀物重悬于 200 µL HBSS 2% FCS 中。</li>
	<li>将重悬的沉淀物转移至新的、已标记的 FACS 管中。</li>
	<li>使用 FACS 检测 T 细胞增殖情况。</li>
	<li>在第 5 天，对 6 孔板中剩余两个孔的细胞重复细胞染色步骤。</li>
</ol>
6. 数据分析
<ol>
	<li>打开“FlowJo”软件，将文件拖入“所有样本”窗口。</li>
	<li>双击第3天采集的未刺激细胞文件，以显示点图，其中纵轴为前向散射光，横轴为侧向散射光。</li>
	<li>点击“多边形门控”，建立设门策略，用于选择淋巴细胞、Thy1.2+ CD3+ 细胞，并区分 CD4+ 和 CD8+ 细胞（见图1）。</li>
</ol>
<img alt="流式细胞术分选图解；FSC-A 对 SSC-A，Thy1.2 对 CD3，CD4 对 CD8a 分析。" class="xfigimg" src="/files/ftp_upload/10503/10503fig1.jpg" data-altupdatedat="1775223722000" data-alt="图 1"> 
图 1：分选策略。 首先根据细胞形态学特征进行分选（左图：FSC-A，SSC-A）。然后对T细胞进行分选（中图：CD3，Thy1.2），并进一步划分为CD4+ T细胞（橙色）和CD8+ T细胞（蓝色）（右图：CD4，CD8）。<a href="https://www.jove.com/files/ftp_upload/10503/10503fig1large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<ol start="4">
	<li>对其他文件重复上述步骤。</li>
	<li>为确定分裂细胞与非分裂细胞的频率，首先点击“Layout editor”以可视化细胞群体。</li>
	<li>将四个管中的 CD4 T 细胞和 CD8 T 细胞拖动至“All sample window”中。</li>
	<li>每个细胞群体对应的图表将显示出来。</li>
	<li>使用直方图展示结果，并选择“CFSE”作为参数，用于比较不同管之间及不同细胞群体之间的差异。</li>
	<li>非分裂细胞维持较高水平的 CFSE，而增殖细胞则将 CFSE 内容分配给子代分裂细胞。</li>
	<li>在非分裂细胞上创建门控（gate），并将其应用于所有管。</li>
	<li>在分裂细胞上创建门控（gate），并将其应用于所有管。</li>
	<li>为分析 CD3+ 细胞中分裂细胞的频率，点击“Table editor”。</li>
	<li>将感兴趣的细胞群体——分裂的 CD8 T 细胞和分裂的 CD4 T 细胞——拖入“Table”中。</li>
	<li>在“Statistic”菜单中选择“frequency of T cells”，然后点击“Create table”，以在新表格中显示频率数据。</li>
	<li>点击“Create table”，频率数值将在新生成的表格中显示。</li>
</ol>

增殖实验常用于免疫学等不同领域，以确定细胞的活化程度。在肿瘤学诊断中，该实验也用于评估患者肿瘤的侵袭性。CFSE染色是一种有效的方法，可用于追踪免疫细胞群体随时间的增殖情况。其他方法可用于细胞周期的表征。BrdU作为一种与CFSE类似的方法，仅在分裂细胞中被掺入。近年来的Fucci小鼠模型甚至无需额外染色即可检测细胞周期的各个时相。

细胞周期分析：利用cfse染色和流式细胞术评估刺激后cd4和cd8 t细胞的增殖

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

来源：Perchet Thibaut1,2,3，Meunier Sylvain1,2,3，Sophie Novault4，Rachel Golub1,2,3 
1 淋巴细胞生成研究组，免疫学系，法国巴黎巴斯德研究所 
2 法国国家健康与医学研究院（INSERM）U1223，法国巴黎 
3 巴黎狄德罗大学，索邦巴黎西岱大学，巴斯德细胞，法国巴黎 
4 流式细胞术平台，细胞计量与生物标志物技术中心（UtechS），转化科学中心，法国巴黎巴斯德研究所
细胞周期是生命的一种普遍过程。在细胞周期中，细胞经历一系列变化，最终分裂为两个子细胞。这一机制会根据生物体的需求，在其整个生命周期中持续发生。通过细胞分裂和胚胎发育，单细胞的受精卵可发育成完整的生物体。在成年阶段，细胞周期在许多关键生物学过程中发挥核心作用，例如组织修复。
细胞分裂的机制是受到严密调控的过程，细胞在最终分裂前会经历一系列有序的改变。尚未进入细胞周期的细胞被称为处于间隙期0（G0）阶段，此阶段的细胞被认为是静止的。当细胞开始进入周期时，可识别出四个明确的阶段：间隙期1（G1）、合成期（S）、间隙期2（G2）和有丝分裂期（M）。G1 期是细胞为DNA合成所需资源进行检查的关卡。随后进入S期，开始DNA复制，接着是G2 间期，这是另一个检查点，用于控制细胞分裂所需的所有关键因素。最后，细胞进入有丝分裂，分裂为两个子细胞。
细胞分裂是许多不同生物系统中一个极具信息价值的参数。在免疫学领域，白细胞增殖的分析可以揭示免疫反应的机制。其他研究领域也依赖于细胞周期分析。例如，在肿瘤发展过程中对细胞周期的分析加深了我们对癌症的理解。
目前已有多种荧光染料可用于追踪细胞增殖。这些染料在化学和光谱特性上各不相同。染料主要分为两类：蛋白质染料通过形成共价键与蛋白质永久结合，而膜染料则通过强烈的疏水相互作用稳定地嵌入细胞膜中。体外和体内免疫细胞增殖的流式细胞术研究是这两类细胞示踪染料最常见的应用之一（1, 2）。
CFSE（羧基荧光素琥珀酰亚胺酯）是一种可标记分裂细胞的荧光染料。最初，所有细胞获得等量的染料；分裂时，细胞将其所含染料平均分配至两个子代细胞中。因此，可通过细胞内染料荧光强度的逐步减弱来追踪细胞周期。CFSE染色后通常结合常规多参数流式细胞术进行检测，该技术是一种高通量、基于荧光的分析方法，可根据细胞的CFSE染色程度实现对其表型和功能的表征（3）。
在以下实验中，我们评估 CD4+ 和 CD8+ T细胞 体外，使用CFSE染色和流式细胞术，在CD3刺激后进行。

在本实验中，我们追踪了脾脏CD4+ 和 CD8+ T 细胞在 体外 培养。3天后，我们未观察到CD4在两种情况下均无明显增殖+ 和 CD8+ 刺激或未刺激的T细胞。这可以在上方图示中观察到。 图2 其中CSFE峰未见降低。然而，5天后我们开始观察到两个群体中均出现增殖，这从CSFE峰的减少中可以明显看出（下图面板， 图2。CFSE 染色清楚地表明，两种 CD4+ 和 CD8+ T细胞在受到刺激后分裂更加活跃。此外，CD8+ T细胞的增殖能力似乎略高于CD4+ 刺激5天后的T细胞
<img alt="流式细胞术图表，显示第3天和第5天CD4+和C...

细胞周期分析：利用CFSE染色和流式细胞术评估刺激后CD4和CD8 T细胞的增殖

来源：Perchet Thibaut1,2,3，Meunier Sylvain1,2,3，Sophie Novault4，Rachel Golub1,2,3  
1 淋巴细胞生成研究组，免疫学系，巴斯德研究所

在大多数免疫学研究中，检测免疫细胞的增殖是一个关键步骤，常用基于CFSE荧光染料的方法进行检测。细胞的正常分裂对免疫细胞至关重要，因为它调控着免疫应答的强度和特异性。例如，T细胞通过增殖来识别并杀伤癌细胞，而B细胞则通过细胞分裂产生特异性抗体。CFSE检测的基本原理是用绿色荧光染料CFSE对细胞进行染色，该染料可进入活细胞并稳定地与胞内蛋白质结合，从而实现永久性标记。因此，当含有染料的亲代细胞分裂时，每个子代细胞将获得亲代细胞一半的荧光强度。该过程在后续的细胞分裂中持续进行，每次分裂后染料的荧光强度逐渐减弱。在达到预期的实验终点时，通过流式细胞术检测每个细胞的荧光强度。这些数据可用于定量分析细胞所经历的分裂次数和分裂模式。如图所示，荧光强度最高的细胞群体来自亲代细胞，第二高的属于子一代细胞，依此类推。峰的数量决定了细胞分裂的次数。此外，如果使用原代免疫细胞，可以将特定的细胞群体（例如T细胞）与CFSE一起用另一种颜色的荧光染料进行标记，并通过多色流式细胞术同时鉴定。这些新数据可绘制在同一张图上，显示具有不同CFSE染色强度的T细胞亚群，从而可特异性地分析T细胞的增殖速率。本视频演示了对小鼠脾细胞进行CFSE染色的实验方案，这些脾细胞经抗CD3抗体刺激后，再进行T细胞标记染色，并通过流式细胞术追踪其细胞增殖情况。首先，穿戴适当的防护服和实验室手套。接着，先用洗涤剂清洗一对镊子和解剖剪，再用70%乙醇冲洗，然后用干净的纸巾擦干。在50毫升离心管中，将1毫升胎牛血清（FCS）与49毫升Hank's平衡盐溶液（HBSS）混合，配制含2%胎牛血清的50毫升HBSS溶液。通过移液器轻柔吹打溶液约10次使其混匀。随后，按照视频方案中所示的FACS分离脾B淋巴细胞的方法，分离小鼠脾细胞。标记四个15毫升离心管，编号为1至4，并向每个离心管中加入1 × 10⁷个分离的脾细胞。接着，向每个离心管中加入3毫升含2% FCS的HBSS。然后，用移液器向每个离心管中加入1微升5微摩尔的羧基荧光素琥珀酰亚胺酯（CFSE）。将离心管置于37°C、5%二氧化碳培养箱中孵育10分钟。1号和2号离心管中的细胞不进行刺激，用于检测脾脏CD4和CD8 T细胞的基础增殖水平。用移液器将10毫升HBSS 2% FCS加入这些试管中。试管三和试管四将用抗CD3抗体进行刺激，以观察其对细胞周期的影响。向试管三和试管四中各加入10毫升HBSS 2% FCS和抗CD3抗体，使抗体终浓度为2.5微克/毫升。接下来，将所有试管在10摄氏度下以370 × g离心7分钟。弃去上清液。将沉淀物重悬于2毫升HBSS 2% FCS中，并将所得溶液分别移入六孔板中的不同孔内。仔细将六孔板标记为1至4号，以便追踪各样品身份。将细胞在37摄氏度、5% CO2条件下孵育三天。第三天，向第一孔和第三孔中各加入 2 毫升 HBSS 2% FCS，这两孔应含有来自第一管和第三管的细胞。用力吹打混匀，然后将样品转移至已标记的 5 毫升 FACS 管中。将六孔板放回培养箱。第二孔和第四孔中剩余的细胞将在第五天进行分析，以研究刺激对细胞周期的长期影响。将 FACS 管在 10°C 下以 370 × g 离心 7 分钟，弃去上清液。随后，向每管中加入 100 微升抗体混合液。将 FACS 管置于冰上避光孵育 20 分钟。接着，向每管中加入 1 毫升 HBSS 2% FCS，并在 10°C 下以 370 × g 离心 7 分钟。弃去上清液。将沉淀物重悬于 200 毫升 HBSS 2% FCS 中，充分混匀。将重悬的沉淀物转移至新的已标记 FACS 管中。然后，使用流式细胞术评估T细胞增殖，具体操作参见FACS实验方案。设门选择淋巴细胞中的CD3阳性细胞，并区分CD4阳性与CD8阳性细胞，记录第一管和第三管的数据。在第五天，对六孔板中剩余两孔的细胞重复细胞染色过程。我们将分析在刺激后第三天和第五天，CD3 刺激对 CD4 和 CD8 阳性细胞细胞周期的影响。首先，点击 FlowJo 图标，并将您的文件拖入“所有样本”窗口。双击第三天采集的未刺激细胞文件，以显示点图，其中纵轴为前向散射光，横轴为侧向散射光。点击“多边形”工具，根据形态学特征圈出淋巴细胞群体。在子群命名窗口中，将该群体命名为“淋巴细胞”，然后点击“确定”。接下来，双击已圈选的群体，在新窗口中将纵轴设为 Thy1.2，横轴设为 CD3。然后，使用“多边形”工具圈出 CD3 与 Thy1.2 双阳性细胞。在新的子群命名窗口中，将该群体命名为“T细胞”，然后点击“确定”。接着，双击已圈选的群体。在新窗口中，将纵轴设为 CD4，横轴设为 CD8。然后，使用“多边形”工具圈出 CD4 阳性细胞群体。在新的子群命名窗口中，将该群体命名为“CD4 T细胞”，然后点击“确定”。现在，使用“多边形”工具圈出 CD8 阳性细胞群体。在新的子群命名窗口中，将该群体命名为“CD8 T细胞”，然后点击“确定”。对其他文件重复上述步骤。为了确定分裂细胞和非分裂细胞的频率，首先点击布局编辑器（Layout Editor）以可视化细胞群体。然后，将四个试管中的CD4 T细胞和CD8 T细胞分别拖动至“所有样本”（All Sample）窗口，相应的细胞群体图表将显示出来。对于每个试管，双击CD8 T细胞的散点图，并在“图形定义”（Graph Definition）中选择直方图，以可视化结果。选择CFSE作为参数，用于比较每个时间点刺激组与未刺激组的细胞群体。非分裂细胞维持较高水平的CFSE，而增殖细胞则将CFSE内容分配给子代分裂细胞。现在，按住 Shift 键的同时，双击直方图。在新窗口中，点击“range”并选择与最高峰对应的 CFSE 范围。在亚群识别窗口中，将该群体命名为“Non-Dividing CD8 T-Cells”，并将另一群体标记为“Dividing CD8 Cells”。接着，重复上述操作，分别在每管中选择分裂和未分裂的 CD4 T 细胞。为分析分裂的 CD3 阳性细胞的频率，请点击“Table Editor”。然后，将感兴趣的群体（Dividing CD8 T-Cells 和 Dividing CD4 T-Cells）拖入表格中。在“Statistic”菜单中选择“Frequency of T-cells”，然后点击“Create Table”，即可在新生成的表格中显示相应的频率数据。

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细胞周期分析:使用CFSE染色和流细胞测定法评估刺激后CD4和CD8 T细胞增殖

tracking t cell division with cfse and flow cytometry

Tracking T cell division with CFSE and flow cytometry shows how immune cells respond after stimulation. The cell cycle is a normal process of life. During it, a cell changes step by step and divides into two daughter cells. This happens throughout life when the body needs new cells for growth and repair.

Cells that are not cycling are in the G0 phase, which means they are quiet. Once a cell enters the cycle, it moves through G1, S, G2, and M phases. G1 is a checkpoint for the resources needed to make DNA. In S phase, DNA replication begins. G2 is another checkpoint before mitosis, when the cell divides into two daughter cells.

Cell division is an important measure in many biological systems. In immunology, leukocyte proliferation can help show how the immune response works. Cell cycle analysis is also useful in tumor research, where it has improved understanding of cancer. Because of this, measuring proliferation is a common part of cell biology studies.

Many fluorescent dyes are used to track cell proliferation. Some dyes are protein dyes that bond permanently to proteins by covalent bonding. Others are membrane dyes that stay in cell membranes through strong hydrophobic interactions. These dyes differ in their chemical and spectral properties, so the choice of dye depends on the experiment.

CFSE, or carboxyfluorescein succinimidyl ester, is a fluorescent dye used to mark dividing cells. At the start, all cells receive the same amount of dye. As cells divide, the dye is split between daughter cells. The drop in CFSE intensity lets researchers follow the cell cycle over time. The stained cells are then analyzed by multi-parametric flow cytometry, a fluorescence-based method that can measure cell features quickly and in detail.

Here, CD4+ and CD8+ T cells are stimulated with CD3 in vitro and their proliferation is assessed with CFSE staining and flow cytometry. This approach helps compare how these two T cell groups divide after activation. It provides a clear way to measure immune cell proliferation in a controlled lab setting.

Tracking T Cell Division with CFSE and Flow Cytometry

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flow cytometry and fluorescence-activated cell sorting (facs): isolation of splenic b lymphocytes

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Sorting B Cells from Spleen with FACS

Flow cytometry and FACS can be used to separate B lymphocytes from a mixed spleen cell sample. The immune system protects the body from infectious organisms and other invaders. White blood cells, also called leukocytes, are the main cells involved in this defense. Leukocytes can be grouped into subpopulations such as myeloid cells, lymphocytes, and dendritic cells. These groups can be distinguished by biological, physical, and functional features. Cell size, granularity, and secretion are...

magnetic activated cell sorting (macs): isolation of thymic t lymphocytes

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Enriching Thymic T Cells with Magnetic Sorting

Thymic T cell enrichment with magnetic sorting helps isolate a targeted immune cell group from a mixed sample. The immune system relies on many cell types, and lymphocytes are a major part of adaptive immunity. B cells usually respond to extracellular infections, such as bacterial infections, while T cells usually respond to intracellular infections, such as viral infections. Magnetic sorting enriches selected cells by using magnetic beads and antibodies against cell surface proteins. First,...

elisa assays: indirect, sandwich, and competitive

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ELISA Methods for Detecting Antigens

ELISA methods detect antigens, antibodies, proteins, hormones, peptides, and other biomolecules in biological samples. Enzyme-linked immunosorbent assay is highly sensitive because it can detect very low antigen levels. It works by using an antigen-antibody interaction, then an enzyme-linked antibody turns a colorless substrate into a colored or fluorescent signal that a plate reader can measure. Different ELISA formats are used for different sample types and antibody setups. Indirect ELISA is...

elispot assay: detection of ifn-&#947; secreting splenocytes

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ELISPOT Assay for Single-Cell Immune Detection

ELISPOT is a sensitive assay for detecting immune cells that release a specific protein. It is based on an ELISA, or enzyme-linked immunosorbent assay, but it shows single-cell responses as visible spots. The method was first described in 1983 by Czerkinsky to count B cell hybridomas that produce antigen-specific immunoglobulins. The assay was later adapted to measure cytokine-producing T lymphocytes. Because of that, ELISPOT is now a gold standard for measuring antigen-specific T cell...

immunohistochemistry and immunocytochemistry: tissue imaging via light microscopy

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Antibody Staining of Tissue and Cells

Antibody staining of tissue and cells lets scientists see where a specific antigen is located. Immunohistochemistry, or IHC, uses thin tissue sections. Immunocytochemistry, or ICC, uses isolated cells or cells grown in culture. Both methods rely on antibodies that carry a chemical or fluorescent tag, such as peroxidase or rhodamine, so the target antigen can be detected under a light microscope. IHC keeps the structure of the whole tissue in place while the sample is stained. That makes it...

antibody generation: producing monoclonal antibodies using hybridomas

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Hybridoma Cell Culture for Monoclonal Antibodies

Hybridoma cell culture is used to produce monoclonal antibodies, or mAbs, with a known antigen specificity. Monoclonal antibodies come from a single clone of cells, so they recognize one target more precisely than polyclonal antibodies, which are a mixed group of antibodies against different antigenic determinants. That difference matters when similar molecules share epitopes, or antigen sites, that can confuse an antibody test. Polyclonal antisera can react with more than one related antigen.

immunofluorescence microscopy: immunofluorescence staining of paraffin-embedded tissue sections

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Fluorescent Antibody Staining of Tissue Slides

Fluorescent antibody staining lets scientists see specific proteins in thin tissue sections on glass slides. It is used to study normal tissue structure and to better understand how disease works. In many cases, the tissue comes from patient biopsies or from experimental in vivo models. The tissue is usually fixed in formalin or paraformaldehyde and embedded in paraffin wax before it is cut. This preservation method supports long-term storage and makes sectioning possible. A microtome is used...

confocal fluorescence microscopy: a technique to determine the localization of proteins in mouse fibroblasts

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Confocal Microscopy for Protein Localization

Confocal fluorescence microscopy is used to show where proteins are located inside cells with better detail than wide-field epifluorescence microscopy. It improves image clarity by scanning the sample with lasers and using a pinhole to block out-of-focus light. This makes it easier to see whether a signal comes from the cell surface or from inside the cell. The method relies on fluorescence, which happens when a molecule absorbs light and then gives off a new photon. A fluorophore is a...

immunoprecipitation-based techniques: purification of endogenous proteins using agarose beads

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Protein Pull-Down with Antibodies and Beads

Immunoprecipitation uses antibodies and Protein A/G agarose beads to isolate a specific protein from a cell lysate. It is also called a pull-down assay. The term "immuno" refers to the antibody, and "precipitation" means pulling a substance out of solution. The target protein can be endogenous or recombinant. Recombinant proteins often carry epitope tags such as myc or flag, which makes purification easier because antibodies against these tags are usually strong and effective. Antibodies...

cell cycle analysis: assessing cd4 and cd8 t cell proliferation after stimulation using cfse staining and flow cytometry

Tracking T cell division with CFSE and flow cytometry shows how immune cells respond after stimulation. The cell cycle is a normal process of life. During it, a cell changes step by step and divides into two daughter cells. This happens throughout life when the body needs new cells for growth and repair. Cells that are not cycling are in the G0 phase, which means they are quiet. Once a cell enters the cycle, it moves through G1, S, G2, and M phases. G1 is a checkpoint for the resources needed...

adoptive cell transfer: introducing donor mouse splenocytes to a host mouse and assessing success via facs

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Tracking Transferred Mouse Splenocytes by FACS

Adoptive cell transfer is a method for moving cells into a host animal to study biology or help treat disease. Researchers can use it to follow cell movement and distribution, or to test how transferred cells behave in a mouse model. Tracking systems such as cell surface markers and CFSE staining can help show where the cells go after transfer. This approach has several uses in mouse studies. It can support cancer research by transferring specific cell populations as an experimental treatment...

assay for cell death: chromium release assay of cytotoxic ability

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Chromium Release Assay for Immune Cell Killing

The chromium release assay measures how well immune cells kill target cells in the lab. It is used to test the cytotoxic ability of T cells, NK cells, and other effector cells against infected cells or tumor cells. This assay works by labeling target cells with chromium-51, a radioactive tracer called Na2 51CrO4. The label quickly marks cellular proteins inside the target cells. When effector cells damage the target-cell membrane, the labeled proteins are released into the culture supernatant...

immunology

该系列涵盖了免疫学实验室的许多主要技术，包括免疫细胞的标签和分类。它还演示了免疫细胞和抗体的增殖方法，以及包括ELISA在内的免疫活性的常见测定方法。最后，演示了免疫组织和细胞样本的染色和成像。

Learn how CFSE staining and flow cytometry track CD4+ and CD8+ T cell division after CD3 stimulation, using cell cycle analysis in the lab.

Tracking T Cell Division with CFSE and Flow Cytometry

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