Name: isolation of cd4+ t-cells and analysis of circulating t-follicular helper (ctfh) cell subsets from peripheral blood using 6-color flow cytometry
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry at JoVE.com

这项工作得到了英国白血病公司向 e t b 和 mja 提供的赠款的支持。

从正常人 (ns) (n = 12) 以及边缘区淋巴瘤 (mzl) (n = 7) 和其他类型的 b 细胞非霍奇金淋巴瘤 (bnhl) (6 例 fl 患者、2例淋巴浆细胞淋巴瘤患者和1个低品位 b 细胞) 中提取血液样本非霍奇金淋巴瘤没有其他指定的病人)。在给予知情、书面同意并对所有研究进行道德认可的情况下, 从莱斯特皇家医务室的血液诊所招募了患者。莱斯特郡、北安普敦郡和鲁特兰研究伦理委员会1获得了道德批准, 病人样本的参考是...

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该协议是一种分析外周血 ctfh 细胞的简单而有效的方法, 能够检测到迄今为止文献中确定的所有相关子集。血液样本可以很容易和有效地获得作为标准门诊诊所的一部分, 和系列样本可以收集平行的临床数据。反过来, 这使得前瞻性研究能够评估 ctfh 亚群作为疾病进展或治疗反应的生物标志物。这些研究将特别需要在疾病中, 其中 tfh 失调与发病机制有关, 如自身免疫和某些类型的固体和血液病癌症?...

t 型滤泡辅助细胞 (tfh) 是 cd4+ t 细胞亚群, 最初的特征是在淋巴组织1中。这些细胞表达 pd-1 和 cxcr5 表面受体, 分泌 il-21 和 il-4, 并显示转录因子 bcl-62,3 的核表达。顾名思义, 它们存在于萌发中心, 对于高亲和力抗体的产生是必不可少的.
失调的 tfh 反应与疾病发病机制有关, 最显著的是自身免疫性疾病, 它们促进自反应 b 细胞4的扩张。它们还在固体5、6 和淋巴癌7的肿瘤微环境中发挥作用。相反, 表面蛋白的遗传缺陷是 tfh 功能所必需的, 如诱导 t 细胞共计器 (icos) 会导致人体免疫缺陷综合征8。人类外周血中的 cd4 + cTfh+细胞被称为循环 t 型滤泡辅助细胞 (ctfh), 被认为是组织 9中 tfh 细胞的记忆室。本文所述方法的目的是分析 cd4 + 细胞分离后的 ctfh亚群。
定义了几个 ctfh 子集, 它们提供 b 细胞帮助的效率因子集而异, 不同的子集为 9、10、11、12.这些子集的相对比例在一些疾病中发生了变化, 最突出的是自身免疫性疾病, 在这些疾病中, 功能较大的 d-1+/ hi 和/或 ctfh2 或 cTfh2 子集的相对增加与较少的亚群相比几乎总是相对增加。功能 pd-1-和/或 ctfh1 子集12。这些变化的程度经常与临床参数有关, 包括疾病活动和自身抗体滴度, 这表明 ctfh 亚系分布作为疾病中的预后生物标志物具有潜在作用, 这可能反映了 tfh 在淋巴组织9,12,13,14。此外, 从参与者身上采集血液样本是快速、安全和可接受的, 因此可以进行连续监测, 以分析疾病进展或对治疗的反应。
与传统外周血单个核细胞 (pbmnc) 悬浮液相比, 分离 cd4+ t 细胞的使用减少了获取大量 ctfh 细胞进行分析所需的时间, 从而实现了更高的吞吐量流式细胞仪实验。当使用流激活单元排序 (facs) 对罕见的 ctfh 子集中的单元格进行排序时, 这尤其有用。为了提高效率, 可以对这些悬浮液进行冷冻保存, 以便在流式细胞术实验中使用样品的 "批处理"。在试验中, 冷冻保存并没有降低 cd4+的纯度。
虽然不同的实验室在发现 ctfh 细胞的早期阶段使用不同的标记对其进行分类, 但这里提出的方法利用了 schmidt 等人提出的两组细胞表面标记的统一方案,15能够在一次流式细胞术实验中同时识别 ctfh 及其9个识别的子集。
由于只使用细胞表面标记物, 细胞不需要固定或渗透, 因此可以保持活力的下游功能研究。这可以通过使用具有相同抗体面板的流式细胞联症进行细胞分类来促进。该面板可以扩展到包括其他标记, 允许使用流式细胞仪的限制。
多色流式细胞术实验的分析可能具有挑战性, 因为在二维点图上的门控具有内在的主观性, 特别是当细胞群在标记荧光中没有一个明确的双模态分布时, 就像ctfh 细胞及其子集的情况。因此, 必须建立有效的控制措施, 以减少文物, 更好地解决民众问题, 并自信地制定门控战略。因此, 在步骤3.4.2 和3.4.3,respectively 中概述了流式细胞仪实验的抗体面板设计和基本控制的设置, 即使用补偿和 fmo 控制。
所有 ctfh 细胞被定义为 cd4+ cTfh+ cTfh-。然后, 可以确定特征 tfh 激活标记 pd-1 的表达水平, 以确定pd-1-、pd-1+或 pd-1 hi ctfh单元的子集。然后, 使用传统的 th1, 2 或17细胞不同表达的趋化因子受体 cTfh 和 ccr6 的组合, ctfh 可以通过 cTfh+ ccr6-, ccr6-, ccr3-ccr6- -, 和 cccr3-ccr6+. 
我们实验室使用的抗体面板显示在表 1中。用户可能需要调整其荧光体的选择, 以考虑其局部流式细胞仪上可用的激光和光过滤器配置。
以下考虑因素影响了荧光的选择。在可能的情况下使用明亮的荧光。特别是, 在最暗 (表达程度较低) 的标记上使用最亮的可用荧光。调光标记包括 pd-1、cxcr3 和 ccr6, 其次包括 cxcr5。我们专门利用较新的 bb、bv 和 buv 荧光, 提供出色的亮度, 从而更容易地解决细胞的不同种群。
尽可能地将荧光体的选择传播到发射光谱中, 以最大限度地减少光谱重叠, 从而降低所需的补偿水平。一个免费的在线工具, 可用于帮助设计流式细胞仪面板可以在这里找到: http://www.bdbioscienes.com/us/s/spectrumviewer。为了节省发射光谱上的空间, 我们使用了一种 "转储通道", 使用的是具有与 apc-h7 (与 cd45ra 结合) 重叠的发射波长的可行染料, 以便能够检测 (和排除) 死亡和/或 cd45ra+ 。单探测器。
本文提出了一种分离外周血 cd4+ t 细胞的方案, 并随后通过流式细胞仪对其进行分析, 以确定不同的和最近描述的循环亚群的比例。

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			<td style="width:179px;">BD Horizon</td>
			<td>562747</td>
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			<td height="20" style="height:20px;width:485px;">BB515 Mouse Anti-Human CD4&#160;Clone &#160;RPA-T4</td>
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			<td>564419</td>
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			<td>560674</td>
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			<td style="width:179px;">BD CompBead</td>
			<td>552843</td>
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			<td height="20" style="height:20px;width:485px;">FACS Aria II Flow Cytometer</td>
			<td style="width:179px;">BD Biosciences</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">FACSDiva 6.1.3</td>
			<td style="width:179px;">BD Biosciences</td>
			<td>643629</td>
			<td>Flow Cytometer Acquisition Software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">FlowJo 10.2</td>
			<td style="width:179px;">Treestar Inc.</td>
			<td style="width:119px;"></td>
			<td>Flow Cytometry Data Analysis Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CXCR3 antibody</td>
			<td style="width:179px;">BD Horizon&#160;</td>
			<td style="width:119px;">565223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CCR6 antibody</td>
			<td style="width:179px;">BD Horizon&#160;</td>
			<td style="width:119px;">563923</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CXCR5 antibody</td>
			<td style="width:179px;">BD Horizon&#160;</td>
			<td style="width:119px;">562747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CD4 antibody</td>
			<td style="width:179px;">BD Horizon&#160;</td>
			<td style="width:119px;">564419</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-PD-1 antibody</td>
			<td style="width:179px;">BD Pharmingen&#160;</td>
			<td style="width:119px;">557946</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CD45RA antibody</td>
			<td style="width:179px;">BD Pharmingen&#160;</td>
			<td style="width:119px;">560674</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Viability Marker</td>
			<td style="width:179px;">ThermoFisher Scientific&#160;</td>
			<td style="width:119px;">L34973</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cd4+ t-cells and analysis of circulating t-follicular helper (ctfh) cell subsets from peripheral blood using 6-color flow cytometry

在自身免疫性条件下检测到 t-滤泡辅助细胞 (tfh) 细胞活性, 当分析 b 细胞非霍奇金淋巴瘤的淋巴结微环境时, 它们的存在与临床结果有关。循环 t-滤泡辅助细胞 (ctfh) 的子集, 血液中 tfh 细胞的循环记忆室, 也在疾病中受到干扰, 因此代表了潜在的新的预测生物标志物。周边血样检测是有利的, 因为它是相对非侵入性的, 并允许简单的串行监测。本文介绍了一种从人的血液中分离 cd4+ t 细胞的方法, 并通过流式细胞仪进一步分析, 以列举 ctfh 细胞及其各种子集的比例 (ctfphd-1-/+/,ctfh1, 1, 17 和 ctfh一个半小时)。然后比较正常受试者和淋巴瘤患者之间这些亚群的水平。我们发现, 这种方法足够稳健, 可以从例行采集的病人材料中获得可靠的结果。我们描述的分析技术可以很容易地适应细胞分类和下游应用, 如 rt-pcr。

cd4+隔离协议可实现高 cd4+纯度, 在我们检测的所有血液样本中都是可靠的 (均为: 966%, sd:2.38, ncos31) (图 3)。
给出了具有代表性的法题 ctfh (cd4+ cTfh+细胞) 的识别方法 (图 4a)。cd4+细胞中 ctfh 细胞的平均值为 29.4% (四分位间范围 (iqr) = 1...

Watch this Scientific Journal Video about Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry at JoVE.com

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

本文介绍了一种从人外周血中分离和表征 cd4 + t 细胞亚群的方法。采用流式细胞仪对纯化的 cd4+ t 细胞进行分析, 以确定 t-滤泡辅助细胞亚群的比例。

cd4+ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

此方法有助于证明在健康状态和患病状态下循环T-卵泡帮助细胞子集之间的差异。该技术的主要优点是速度快，样品可以批量处理，并且细胞保持活力，用于下游功能分析。这项技术有助于评估循环的T-卵泡帮助器子集作为疾病进展的生物标志物，因为它们与临床严重性标志物相关。帮助演示这个程序将是露西娅·皮博士

Research

JoVE Journal

Biology

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

科研

生物学

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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 ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

人外周血代文化血液的产物内皮细胞

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Isolation and Characterization of Microvesicles from Peripheral Blood

从外周血中分离和鉴定的微泡

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

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 ...