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: 2019-01-07T14:00:00.000+00:00
Duration: 7 min 39 s
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 0q250. i 122, 卫生研究局 (hra) 委员会东 midlands-devce, 参考14/em/176正常科目。
1. 从全外周血中分离 cd4+ t 细胞
<ol><li>通过标准静脉穿刺和工艺, 尽快将新鲜外周血(2 至15毫升) 放入 k2 edta 管中, 以保持最大的活力。 请注意:使用标准的实验室个人防护设备 (外套、手套、眼镜), 并在二级生物安全柜中开展工作。</li>
	<li>将血液和所有必要的试剂 (cd4+浓缩鸡尾酒、2% 的胎牛血清磷酸盐缓冲盐水 (fbsbsp)、密度梯度培养基和冷冻介质 (如果冷冻保存细胞) 带到室温。</li>
	<li>将血液与市售的抗体混合物混合, 以丰富人类 cd4+ t 细胞。每1毫升血液使用50μl 试剂。使用50毫升锥形聚丙烯管5至15毫升的血液。 请注意:如果使用2至4毫升的血液, 请在14毫升锥形聚丙烯管中执行此步骤。</li>
	<li>在室温下将混合物生也就是20分钟。</li>
	<li>用相同体积的 2% fbse/pbs 稀释混合物</li>
	<li>
慢慢地将这种稀释的混合物分层在密度梯度介质的顶部, 以避免干扰界面, 并立即进行离心, 以避免血液扩散到密度梯度介质中。
	<ol>
<li>对于2至3毫升的血液, 在14毫升锥形聚丙烯管中使用3毫升密度梯度介质, 但对于4毫升的血液, 在 14 ml 锥形聚丙烯管中使用4毫升密度梯度介质, 对于5至 15 ml 血液, 在50毫升锥形聚丙烯管中使用15毫升密度梯度介质。</li>
	</ol>
</li>
	<li>在 1200 x 克的温度下离心层状混合物 20分钟 , 在20°c 下关闭制动器。 请注意:环境温度低于环境可能会导致红细胞和粒细胞的污染。较低的速度将导致 cd4+隔离过程失败。离开休息将减少细胞产量。使用工作台顶部离心机与转子, 可以关闭, 以避免气溶胶。</li>
	<li>使用巴斯德移液器从界面中取出浓缩的 cd4+细胞层。 请注意:浓缩的细胞层将类似于白色多云细胞的标准 "蓬松涂层", 但由于只有 cd4+细胞存在, 这自然会更小, 更难看到。</li>
	<li>在 cd4+细胞中添加2% 的 fbsp, 总体积不超过10毫升, 然后用2% 的 fbsp 清洗两次, 每次清洗时以 400 x 克的速度离心 10分钟 。</li>
	<li>将颗粒重新放在2% 的 fbssp 中, 并对染色的细胞进行计数, 以确定细胞数量和活力。</li>
	<li>可选步骤: 在1毫升的 aliquots 中,在冷冻介质中重新悬浮 5 x 10 5 至 1 x 10 6 细胞 (fbs 中的二甲基亚硫酸为 10%)。 请注意:某些表面标记的水平可能会通过低温保存和解冻来改变。因此, 对所有样品进行一致处理是非常重要的。为了最大限度地减少分析变量的差异, 本研究对样品进行了冷冻保存和配料分析。</li>
</ol>2. 流式细胞仪
<ol><li>在37°c 的水浴中快速解冻冷冻保存的 cd4+细胞, 并在预热介质中清洗一次 (rpmi 1640 + l-谷氨酰胺补充 10% fbs 和1x 青霉素链霉素)。</li>
	<li>将细胞加入9毫升的培养基中, 离心剂在 400 x克处 5分钟, 并去除上清液。</li>
	<li>在 pbs (50μl) 中重新移植1% 的牛血清白蛋白 (bsa) 中的细胞, 以便每个要测试的条件使用 5 x10 5和 1 x 106 的细胞。</li>
	<li>将细胞与染色缓冲液 (50μl) 混合。 注: 由于两种或两种染料固有的化学性质, 同时使用两个或两个以上的 bv 或 buv 污渍时, 明亮的污渍缓冲可防止可能干扰数据分析的各种染色制品。在单一或未染色条件下, 50μl 的明亮染色缓冲液可替代50μl 的 1% bsa/pbs。</li>
	<li>将抗体添加到细胞中 (根据表1中的 "使用量" 列), 在黑暗中在冰上孵育30分钟。</li>
	<li>每次清洗时, 以 600 x 克的速度离心 3分钟, 在 1% bsa/pbs 中清洗细胞两次。</li>
	<li>在 1% bsa/pbs (400μl) 中重新移植并转移到流式细胞仪采集管中。</li>
	<li>在获取流式细胞仪上的数据之前, 将细胞轻轻涡旋:</li>
</ol>3. 流式细胞仪控制和设置
<ol><li>使用未染色的样本, 设置光电二极管电压, 以便淋巴细胞可以从明显的碎片和死细胞 (具有高侧向散射 (ssc) 区域和低向前散射 (fsc) 区域的事件)16。使用单个染色样品, 设置光电倍增管 (pmt) 电压, 以便从背景荧光中识别出正荧光, 同时确保所有事件都在可检测的范围内。 请注意:改进的方法是使用模糊荧光磁珠绘制每个 pmt 电压的 cv 图, 以找到最佳分辨率的最小电压 ("峰值 2" 法19)。</li>
	<li>通过使用带有捕获珠的单个污渍生成补偿矩阵来考虑光谱重叠。在 1% bsa/pbs (100μl) 和涡旋中加入市售补偿珠 (60μl) 和负控制珠 (60μl)。 请注意:所使用的捕获珠子必须与所使用抗体的宿主物种和 igg 同型相匹配。如果任何串联染料的批号发生变化, 则应重新计算补偿矩阵, 因为它们在批次之间的发射光谱会显示出相当大的变化。</li>
	<li>加入单一抗体 (20μl) 和涡旋。</li>
	<li>在室温下在黑暗中孵化30分钟。</li>
	<li>以 200 x克离心 10分钟, 并丢弃上清液。</li>
	<li>在 1μp bs (500μl) 和涡旋中进行再利用。</li>
	<li>根据制造商的说明, 使用采集软件中指定的补偿矩阵发生器使用流式细胞仪采集样品。</li>
	<li>使用 fmo 控件来指导正标记荧光的门的放置, 以说明染料溢出的原因, 这是使用≥4色16的实验中背景荧光的主要来源。按照步骤3中描述的一般细胞表面染色协议, 但对于每个条件, 省略染色步骤中的一个荧光 (其中省略 cd45ra, 也省略活/死染色)。每种情况下获得 100, 000个淋巴细胞。</li>
	<li>将栅极阳性率设置为≤0.5% 的细胞。有关 fmo 控件的示例, 请参见图 2 。</li>
</ol>4. 数据分析
<ol><li>使用流式细胞仪的采集软件, 在 fsc 参数上设置 5, 000个单位的阈值, 以排除非常小的碎片。</li>
	<li>使用停止门获取 10, 000个 ctfh 细胞 (cd4+ cd45ra-cxcr5+). 请注意:单个用户可以收集 10, 000多个单元格。这个数字是在收集足够的单元格以提供有意义的结果和收集所需的时间之间的妥协。</li>
	<li>使用 fsc-parta/ssc 面积点图, 绘制一个椭圆或多边形门, 以选择淋巴细胞的数量, 同时不包括碎片和公开死亡细胞 (ssc-a 区高、fsc 面积低的事件)。</li>
	<li>使用 fsc-fore/fsc-gow 点图, 绘制多边形门选择单个单元格, 同时排除双单元 (双面具有增加的面积, 但宽度与单个单元格相似)。</li>
	<li>使用 fsc 面积/cd45ra 和活力标记点图, 绘制一个矩形门选择活的 cd45ra-细胞(细胞具有低荧光为这个标记)。</li>
	<li>使用 fsc 面积/cd4 点图, 绘制一个矩形门, 以选择 cd4+细胞 (此标记具有高荧光的细胞)。</li>
	<li>使用 cd4/cxcr5 点图, 绘制矩形门选择 cTfh+ (即 ctfh) 细胞 (此标记具有高荧光的细胞)。</li>
	<li>使用 cxcr3/ccr6 点图, 放置一个四门将 ctfh 细胞细分为 ctfh1, 2 和17单元 (每个标记的单元数都是高和低的)。 请注意:表型 cd4+ cxcr5+ cxcr3+ ccr6+的细胞特征较差。我们将这些细胞称为 ctfh带18,因为在 th17 和 th1 之间过渡的常规辅助 t 细胞(cd4+ cxcr5-) 显示了 cxcr3 和 ccr6 的表达, 并被描述为 ctfh一个半小时.</li>
	<li>使用 pd-1 直方图, 使用范围工具将 ctfh 细胞 (或如果愿意, 将单个 ctfh1, 17 或-17 子集) 细分为 pd-1-/+或hi人群。 请注意:第 d-1+和 pd-1hi 之间的区别在文献中没有得到很好的界定。阈值被设定为相同的 pd-1 强度所需的检测真正的 t直肠癌淋巴细胞悬浮液使用流式细胞仪与相同的抗体面板。或者, 可以将 icos 的标记添加到抗体面板中, 因为只有 pd1hi 细胞是 icos +。</li>
</ol>

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作者没有什么可透露的。

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。此外, 本方案仅使用细胞表面标记, 通过流式细胞术对 ctfh 细胞进行分选, 可以研究疾病中 ctfh 功能的变化。对 mzl 患者的 ctfh 细胞进行排序, 使我们能够发现与正常受试者21相比, 基因表达谱存在差异。
我们发现, 有效的 cd4+ t 细胞分离改善了数据分析。我们详细介绍了所涉及的步骤, 因为离心机制动和速度等小问题对隔离过程至关重要。另一个重要步骤, 所有流式细胞术分析, 是设置补偿和 fmo 控制。
pd-1 在 ctfh 细胞上的表达各不相同, pd-1 最高的细胞可能是最有效的, 因此也是相关的, 特别是在自身免疫性疾病11,12的研究中。一个重要的问题是确定确定 pd-1hi 单元的大门, 即激活的 ctfh 子集, 因为文献中没有界定标准限制。这个重要但较小的子集可能反映了淋巴组织11中的活性 tfh 分化.为了克服这一挑战, 设定了阈值, 其设定与用相同的抗体面板从人体扁桃体中检测真正 tfh 细胞所需的门槛相同。我们认识到, 获得扁桃体可能是一些用户的问题。另一种方法是通过添加抗 icos 来扩大本协议中使用的抗体面板, 因为只有 d-1hi 细胞是 icos + 12。
在这里, 我们提出了一个抗体面板, 以检测表面标记特征循环 cd4+ t 细胞。循环 t-滤泡调节细胞 (ctfr) 是 t-滤泡调节细胞 (tfr) 的血液记忆室, 存在于淋巴组织中, 在调节萌发中心反应 22, 23 中具有重要作用.ctfr 是血液 cd4+ cTfr+细胞, 共表达 t 调控细胞标记物, 包括转录因子 foxp3。在 cTfr 旁边测量 ctfr 可以提供更完整的生殖中心活动图片, 并可以在疾病中呈现一个生物标志物本身 24.虽然 ctfr 的数量本质上是低的 (平均: 1.82, sd:1.40, n = 我们自己的实验中 ctfr 与 cTfr分离ctfr 也会增加 cTfr 分析的特异性。在我们的面板 (如 cd25 和 cd12725)中添加特定 t-监管细胞表面标记的组合, 可以使用非常相似的协议检测到 ctfr 以及同一实验中的所有 cTfr 子集。或者, 可以使用更经典的细胞内调节标记 ffmp3, 虽然这需要固定和渗透, 防止下游功能检测。然而, 在专注于 ctfh 分析时, 定义一个最小面板也有好处, 该面板可与标准实验室流式细胞仪结合使用, 以获得临床或实验中有用的结果。

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。

<table><tbody><tr><td>RosetteSep Human CD4+ T Cell Enrichment Cocktail</td><td>STEMCELL TECHNOLOGIES</td><td>15062</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare Life Sciences</td><td>17144003</td><td></td></tr><tr><td>BUV395 Mouse Anti-Human CD183&amp;nbsp;Clone 1C6/CXCR3</td><td>BD Horizon</td><td>565223</td><td></td></tr><tr><td>BV711 Mouse Anti-Human CD196 (CCR6)&amp;nbsp;Clone 11A9</td><td>BD Horizon</td><td>563923</td><td></td></tr><tr><td>BV421 Rat Anti-Human CXCR5 (CD185)&amp;nbsp;Clone&amp;nbsp;RF8B2</td><td>BD Horizon</td><td>562747</td><td></td></tr><tr><td>BB515 Mouse Anti-Human CD4&amp;nbsp;Clone &amp;nbsp;RPA-T4</td><td>BD Horizon</td><td>564419</td><td></td></tr><tr><td>PE Mouse Anti-Human CD279&amp;nbsp;Clone MIH4</td><td>BD Pharmingen</td><td>557946</td><td></td></tr><tr><td>APC-H7 Mouse Anti-Human CD45RA Clone&amp;nbsp; HI100</td><td>BD Pharmingen</td><td>560674</td><td></td></tr><tr><td>LIVE/DEAD Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitation</td><td>ThermoFisher Scientific</td><td>L34973</td><td></td></tr><tr><td>Brilliant Stain Buffer</td><td>BD Horizon</td><td>563794</td><td></td></tr><tr><td>Anti-Mouse Ig, &amp;kappa;/Negative Control Compensation Particles Set</td><td>BD CompBead</td><td>552843</td><td></td></tr><tr><td>FACS Aria II Flow Cytometer</td><td>BD Biosciences</td><td>644832</td><td></td></tr><tr><td>FACSDiva 6.1.3</td><td>BD Biosciences</td><td>643629</td><td>Flow Cytometer Acquisition Software</td></tr><tr><td>FlowJo 10.2</td><td>Treestar Inc.</td><td></td><td>Flow Cytometry Data Analysis Software</td></tr><tr><td>Anti-CXCR3 antibody</td><td>BD Horizon&amp;nbsp;</td><td>565223</td><td></td></tr><tr><td>Anti-CCR6 antibody</td><td>BD Horizon&amp;nbsp;</td><td>563923</td><td></td></tr><tr><td>Anti-CXCR5 antibody</td><td>BD Horizon&amp;nbsp;</td><td>562747</td><td></td></tr><tr><td>Anti-CD4 antibody</td><td>BD Horizon&amp;nbsp;</td><td>564419</td><td></td></tr><tr><td>Anti-PD-1 antibody</td><td>BD Pharmingen&amp;nbsp;</td><td>557946</td><td></td></tr><tr><td>Anti-CD45RA antibody</td><td>BD Pharmingen&amp;nbsp;</td><td>560674</td><td></td></tr><tr><td>Viability Marker</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>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) = 10.8), 在12个正常人中。多项不同疾病的研究, 包括肝细胞癌17,20, 系统性红斑狼疮11,21, 类风湿关节炎11,14在检测健康控制和患者之间的整体 ctfh 差异的能力上存在冲突。正常人与 mzl 或 bnhl 患者的总 ctfh 没有明显差异 (图 4b)21。
图 5a 显示了具有代表性的正常人和 bnhl 患者在 ctfh 细胞内的 pd-1 表达的识别, 以便进行比较。mzl 和 bnhl 患者的 pd-1 表达明显高于正常患者 (图 5b)21。在多种自身免疫性疾病11,12,13, 14中也显示了类似的 pd-1 表达的增加。
图 6a 显示了利用具有代表性的正常主体的 cxcr3 和 ccr6 表达式在 ctfh 细胞群中识别 ctfh1, 2, 17 和半。mzl 和 bnhl 患者的 ctfh1 细胞比例明显高于正常患者 (图 6b)21。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58431/58431fig1.jpg"> 图 1* 说明用于识别 ctfh 细胞及其子集的总体门控策略.从左上角, 淋巴细胞的数量被区分, 和双细胞被排除在外。实时 cd45ra-使用 "转储通道" 选择单元格。cd4+细胞被门控, cTfh+细胞被识别为 ctfh。ctfh 使用 cTfh 和 ccr6 表达式分为 ctfh1、2或17-like。然后区分这些子集中的 d-1 表达式。从具有代表性的正常人体血液样本中提取的数字。fsc-a, fsc 面积;ssc-a ssc 领域;fsc-w, fsc 宽度。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58431/58431fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58431/58431fig2.jpg"> 图 2:fmo 控制的说明.每个双轴流式细胞仪图显示 cd4+细胞染色与所有的荧光, 除了一个感兴趣的证明最小的门荧光阳性可以设置。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58431/58431fig2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58431/58431fig3.jpg"> 图 3:显示 cd4 识别的双轴流式细胞仪图+细胞门控后活的淋巴细胞.从具有代表性的正常主体血液样本中提取。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58431/58431fig3large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58431/58431fig4.jpg"> 图 4: ctfh 细胞的鉴定.(a) 双轴流式细胞术图显示 cxcr5+在 cd4+ cd45ra门控的细胞上的表达。取自一个有代表性的正常主题。(b) ctfh在正常受试者 cd4 + 细胞总数中的相对百分比 (n = 12)、mzl (n = 7)、bnhl (n = 9)。水平线表示中值, 误差线表示四分位数之间的范围。使用曼恩-惠特尼 u 测试的组之间没有发现显著差异。这一数字已从 byford 等人21人修改。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58431/58431fig4large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58431/58431fig5.jpg"> 图 5: pd-1 表达和与 ctfh 细胞的关系.(a) 流式细胞仪直方图, 用于确定总 ctfh 细胞中的 pd-1 表达。从一个有代表性的正常人和 bnhl 患者中提取。(b) d-1+ 细胞占 ctfh 细胞总数的比例。水平线表示中值, 误差线表示四分位数之间的范围。正常人之间的介质 (曼-惠特尼 u-测试) 有显著差异 (21.5%, iqr = 10.8, n = 12) 和淋巴瘤患者 (mzl 54.1%, iqr = 21.5, n = 7, p = 0.0008 和 bnhl 45.2%, iqr = 11.4, n = 9, p = 0.0008)。这一数字已从 byford 等人21人修改。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58431/58431fig5large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58431/58431fig6.jpg"> 图 6: cxcr3 和 ccr6 表达式及其与 ctfh1 数字的关系.(a) 双轴流式细胞术图显示 cxcr3 和 ccr6 在 cd4 + cd45ra-cxcr5+细胞上的表达. 从一个有代表性的正常人和 bnhl 患者中提取。(b) ctfh1 细胞占 ctfh 细胞总数的百分比。水平线表示中值, 误差线表示四分位数之间的范围。正常人间的介质 (曼-惠特尼 u-测试) 有显著差异 (20.8%, iqr = 6.7, n = 12) 和淋巴瘤患者 (mzl 32.1%, iqr = 6.8, n = 7, p = 0.013, bnhl 30.4%, iqr = 7.6%, n = 9, p = 0.0056)。这一数字已从 byford 等人21人修改。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58431/58431fig6large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/58431/58431table1.jpg"> 表 1: 流式细胞仪抗体面板。

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

isolation-cd4-t-cells-analysis-circulating-t-follicular-helper-ctfh

Universidad San Sebastian

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

12.1K 次观看.  University of Leicester. 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

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

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

从Sézary综合征患者中分离人外周血单核细胞和CD4 + T细胞以进行转录组学分析

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

科研

癌症研究

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

免疫与感染

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

在预处理肿瘤细胞的同种异体共培养环境中分析人 T 细胞活性

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

双氟铬流式细胞仪对七种免疫细胞子集的判别

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

使用微泡从人外周血单核细胞样品中分离、活化和扩增基于浮选的 T 细胞

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

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

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

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

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

教育

JoVE Science Education

高级生物学

免疫学

An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers

Source: Steblyanko, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58143">Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro technique to study immune synapse formation between primary human T cells and supported lipid bilayers (SLB). T cell surface proteins interact with respective ligands immobilized on SLB to form an immune synapse, followed by the movement of lytic granules towards the synapse, which is visualized using fluorescence microscopy.

一种研究 T 细胞与支持的平面脂质双层相互作用的体外技术

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of CD4+ T Cells for an ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Selective Isolation of CD4+ V&delta;1+ &gamma;&delta; T Cells from Human Peripheral Blood Mononuclear Cells

Source: Welker, C., et al. Isolation and Ex Vivo <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53482">Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor.</a> J. Vis. Exp. (2015).
This video demonstrates a protocol for the magnetic labeling-based isolation of a low-concentration population of CD4+V&#948;1+&#947;&#948; T cells from human peripheral blood mononuclear cells (PBMCs). Using a combination of FITC-conjugated anti-V&#948;1 antibodies, magnetic anti-FITC microbeads, and anti-CD4 antibody-coated superparamagnetic beads, the CD4+V&#948;1+ T-cells can be successfully purified from the PBMC population.

从人外周血单核细胞中选择性分离 CD4+ Vδ1+ γδ T 细胞

Source: Welker, C., et al. Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor. J. Vis. Exp. ...

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

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