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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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+TFH+Cell+Function+and+Increased+TFR+Cells+Contribute+to+Defective+Antibody+Production+in+Aging.">Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.</a> Cell Reports. 12 (2), 163-171 (2015).</li><li>Yu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+CD25+CD127low/-+T+Cells:+A+More+Specific+Treg+Population+in+Human+Peripheral+Blood.">CD4+CD25+CD127low/- T Cells: A More Specific Treg Population in Human Peripheral Blood.</a> Inflammation. 35 (6), 1773-1780 (2012).</li></ol>

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?...

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>17144003</td>
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			<td>565223</td>
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			<td height="20" style="height:20px;width:485px;">BV711 Mouse Anti-Human CD196 (CCR6)&#160;Clone 11A9</td>
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			<td>563923</td>
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			<td height="20" style="height:20px;width:485px;">BV421 Rat Anti-Human CXCR5 (CD185)&#160;Clone&#160;RF8B2</td>
			<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 height="20" style="height:20px;width:485px;">PE Mouse Anti-Human CD279&#160;Clone MIH4</td>
			<td style="width:179px;">BD Pharmingen</td>
			<td>557946</td>
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			<td height="20" style="height:20px;width:485px;">APC-H7 Mouse Anti-Human CD45RA Clone&#160; HI100</td>
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			<td>560674</td>
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			<td height="21" style="height:21px;width:485px;">LIVE/DEAD Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitation</td>
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			<td height="20" style="height:20px;width:485px;">Anti-Mouse Ig, &#954;/Negative Control Compensation Particles Set</td>
			<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>
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			<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>
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			<td height="20" style="height:20px;width:485px;">FlowJo 10.2</td>
			<td style="width:179px;">Treestar Inc.</td>
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			<td>Flow Cytometry Data Analysis Software</td>
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			<td height="21" style="height:21px;width:485px;">Anti-CXCR3 antibody</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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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

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

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

12.0K 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

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

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

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

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

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

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 unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

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

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

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 ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

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 ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

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

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 the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

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 ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

流细胞测定方法测量造血干细胞和T细胞的线粒体质量及膜电位

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.