Name: analysis of t-cell receptor-induced calcium influx in primary murine t-cells by full spectrum flow cytometry
Uploaded: 2022-12-16T13:25:00
Description: Watch this Scientific Journal Video about Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry at JoVE.com

R01AI132419， CU |AMC ImmunoMicro流式细胞术共享资源，RRID：SCR_021321，非常感谢Cytek的同事在Aurora和SpectroFlo软件上不断讨论全光谱细胞术分析。人物是用 BioRender.com 创造的。

根据IACUC协议在科罗拉多大学安舒茨医学院院区维持小鼠。所有小鼠均根据AAALAC标准实施安乐死。
1.小鼠脾脏免疫细胞的制备
注意：用CO2 安乐死对幼稚小鼠实施安乐死。从杰克逊实验室购买并在内部繁殖的C57BL / 6小鼠用于6-12周龄的实验。雄性和雌性小鼠都用于实验。
<ol><li>用70%乙醇消毒小鼠皮肤，以减少外部污染物进入样品的可能...

<ol>
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该协议描述了一种优化的测定方法，旨在使用全光谱流式细胞术7，8测量加载滴定Indo-1比例指示剂染料的原代鼠T细胞中的钙反应。使用全光谱流式细胞术进行钙通量测定的优点是能够结合Indo-1荧光评估对表面细胞标志物进行多重染色。全光谱流式细胞术的优点是允许使用高度重叠的染料，从而增加可在组合中使用的标记物数量。这是因为全光谱流式细?...

T细胞受体（TCR）诱导的钙内流是T细胞活化的有用指标，通常用于确定T细胞群在TCR信号通路1的近端步骤中是否具有受损的反应。钙内流的测量通常通过用一种或一对荧光钙指示剂染料预先标记T细胞来进行，然后在TCR交联2，3，4后使用流式细胞术实时检查荧光信号。Indo-1是一种比例法钙染料，由紫外激光激发，在取决于钙结合5的两个不同波长处具有峰值发射，并且是流式细胞术分析活淋巴细胞中钙反应的常用指示染料。由于Indo-1的发射曲线相当广泛，因此将Indo-1评估与通过带通流式细胞术同时分析多个细胞表面标志物相结合可能具有挑战性。这种限制限制了流式细胞术对预纯化的T细胞群或由一组有限的细胞表面分子鉴定的细胞群的钙反应分析的使用。
为了解决使用带通流式细胞术测量原代淋巴细胞异质群体的钙反应的局限性，开发了一种使用全光谱流式细胞术测量Indo-1荧光的方案。该方法允许利用全光谱流式细胞术的高度灵活性，将Indo-1与针对细胞表面分子的抗体组合进行多重分析。与传统流式细胞术相比，使用全光谱流式细胞术的优势在于它能够区分荧光信号和高度重叠的染料，从而增加每个样品中可同时评估的表面标志物的数量。常规流式细胞术使用带通滤光片，并且每个检测器系统限制为一种荧光染料6。全光谱流式细胞术使用五激光光谱流式细胞术系统7，8上的64个检测器收集荧光染料整个光谱的信号。此外，全光谱流式细胞术利用了APD（雪崩光电二极管）检测器，与传统流式细胞仪8上的光电倍增管检测器相比，APD（Avalanche 光电二极管）检测器的灵敏度更高。因此，这种方法是异质细胞群的理想选择，例如外周血单核细胞或鼠次级淋巴器官细胞悬液，因为它消除了在钙染料标记之前分离特定T细胞群的需要。相反，数据收集后的细胞表面标志物表达谱和流式细胞术门控可用于评估每个目标群体中的钙反应。如本报告所示，Indo-1 可以很容易地与八种荧光染料偶联抗体结合，从而产生总共 10 种独特的光谱特征。此外，该方法可以很容易地应用于来自同源不同小鼠系的细胞混合物，允许与来自基因靶向小鼠系的细胞相比同时分析野生型T细胞中的钙反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>12 well TC treated plates</td>
			<td>Cell Treat</td>
			<td>229111</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical</td>
			<td>Greiner Bio1</td>
			<td>41-12-17-03</td>
			<td>50 mL Polypropylene centrifuge tubes with cap</td>
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		<tr>
			<td>5mL polysterene flow tubes</td>
			<td>Corning</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL syringe</td>
			<td>BD syringe</td>
			<td>309646</td>
			<td>plunger only is used sheith is discarded</td>
		</tr>
		<tr>
			<td>70uM filter</td>
			<td>Greiner bio1</td>
			<td>542070</td>
			<td></td>
		</tr>
		<tr>
			<td>aCD3 (17A2)</td>
			<td>Biolegend</td>
			<td>100202</td>
			<td></td>
		</tr>
		<tr>
			<td>AKC lysis Buffer</td>
			<td>Gibco</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>Aurora Spectral Flowcytometer</td>
			<td>https://cytekbio.com/pages/aurora</td>
			<td></td>
			<td></td>
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			<td>Bath Beads</td>
			<td>coleparmer</td>
			<td>Item # UX-06274-52</td>
			<td></td>
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			<td>CD19 PE</td>
			<td>Tonbo</td>
			<td>50-0193-U100</td>
			<td></td>
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		<tr>
			<td>CD1d Tetramer APC</td>
			<td>NIH</td>
			<td></td>
			<td></td>
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			<td>CD25 PECy7</td>
			<td>ebioscience</td>
			<td>15-0251</td>
			<td></td>
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			<td>CD4 APC Cy7</td>
			<td>Tonbo</td>
			<td>25-0042-U100</td>
			<td></td>
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			<td>CD8a FITC</td>
			<td>ebioscience</td>
			<td>11-0081-85</td>
			<td></td>
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		<tr>
			<td>Cell Incubator</td>
			<td>Formal Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tools forceps</td>
			<td>McKesson</td>
			<td>#487593</td>
			<td>Tissue Forceps McKesson Adson 4-3/4 Inch Length Office Grade Stainless Steel NonSterile NonLocking Thumb Handle 1 X 2 Teeth</td>
		</tr>
		<tr>
			<td>Dissection Tools Scissors</td>
			<td>McKesson</td>
			<td>#970135</td>
			<td>Operating Scissors McKesson Argent&#8482; 4-1/2 Inch Surgical Grade Stainless Steel Finger Ring Handle Straight Sharp Tip / Sharp Tip</td>
		</tr>
		<tr>
			<td>DPBS 1x</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td>DPBS&#160;</td>
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			<td>EGTA</td>
			<td>Fisher</td>
			<td>NC1280093</td>
			<td></td>
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			<td>FBS</td>
			<td>Hyclond</td>
			<td>SH30071.03</td>
			<td>lot AE29165301</td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>https://www.flowjo.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indo1-AM Ester Dye</td>
			<td>ebioscience</td>
			<td>65-085-39</td>
			<td>Calcium Loading Dye&#160;</td>
		</tr>
		<tr>
			<td>ionomycin</td>
			<td>Millipore</td>
			<td>407951-1mg</td>
			<td></td>
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		<tr>
			<td>Live/Dead Ghost 540</td>
			<td>Tonbo</td>
			<td>13-0879-T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.7mL</td>
			<td>Light Labs</td>
			<td>A-7001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/L-Glutamine</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>PRN694</td>
			<td>Med Chem Express</td>
			<td>Hy-12688</td>
			<td></td>
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		<tr>
			<td>Purified Anti-Mouse CD16/CD32 (FC Shield) (2.4G2)</td>
			<td>Tonbo</td>
			<td>70-0161-M001</td>
			<td>FC Block</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>1875093</td>
			<td>&#160;+ phenol red</td>
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		<tr>
			<td>RPMI phenol free</td>
			<td>Gibco</td>
			<td>11835030</td>
			<td>&#160;-phenol red</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra612</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR&#946; PerCP Cy5.5</td>
			<td>ebioscience</td>
			<td>45-5961-82</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR&#947;/&#948; Pe Cy5</td>
			<td>ebioscience</td>
			<td>15-5961-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Vi-Cell Blu Reagent Pack</td>
			<td></td>
			<td>Product No: C06019</td>
			<td>Includes Tripan</td>
		</tr>
		<tr>
			<td>Vi-Cell Blu</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Fisher Brand Dry bath</td>
			<td></td>
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analysis of t-cell receptor-induced calcium influx in primary murine t-cells by full spectrum flow cytometry

响应 T 细胞受体刺激的钙内流是 T 细胞信号传导的常见指标。已经开发了几种钙指示剂染料，用于通过带通流式细胞术评估钙信号传导。该协议旨在使用全光谱流式细胞术测量原代鼠T细胞中的钙反应。用比例钙指示染料Indo-1标记总脾细胞，以及一组针对细胞表面分子的荧光染料偶联抗体。利用全光谱流式细胞术的功能，为将各种细胞表面染色剂与Indo-1结合使用提供了一个平台。然后在加入抗CD3抗体以刺激T细胞受体之前和之后在37°C下实时分析细胞。在解混合光谱信号后，计算钙结合与无钙Indo-1的比率，并且可以随着时间的推移为每个门控的脾细胞群可视化。该技术可以同时分析多个细胞群中的钙反应。

使用具有多重表面染色剂的Indo-1比例染料评估原代鼠T细胞中钙反应的实验工作流程如图 1所示。在收获小鼠脾细胞并将其处理成单细胞悬液后，用Indo-1AM酯染色细胞并用荧光染料连接的抗体染色表面。一旦染料上样和抗体染色完成，淋巴细胞样品被加热至生物温度（37°C）。然后使用全光谱流式细胞术对Indo-1荧光进行分析，在对所需的单个细胞类型进行门控后，无需在分析前...

Watch this Scientific Journal Video about Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry at JoVE.com

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

钙内流是T细胞信号传导的量度，是分析对T细胞受体刺激反应的有效方法。该协议将Indo-1与针对细胞表面分子的抗体组合多重化，利用了全光谱流式细胞术的高度灵活性。

全谱流式细胞术分析原代小鼠T细胞受体诱导的钙内流

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...

该协议允许研究人员同时测量原代T细胞中的钙反应，同时评估其亚群和表面表型。这种技术的主要优点是它避免了在检查其钙反应之前分离特定细胞亚群的需要。首先，通过将 50 微升 DMSO 添加到含有 50 微克 Indo-1 AM 的小瓶中，制备每微升 1 微克的 Indo-1 AM 储备溶液。将储备溶液稀释成适当体积的cRPMI，浓度为每毫升6微克。将培养基放入 37 摄氏度的水浴中。然后使用该培养基以一比一的比例稀释先前制备的细胞悬液，以获得每毫升5至600万个细胞。接下来，向12孔组织培养板中，每个实验条件每孔加入一毫升细胞悬液。通过将EGTA添加到Indo-1染料负载细胞中，制备用于无钙Indo-1的单个染色对照样品。准备一个没有荧光团或Indo-1染料的生物阴性对照孔。通过在流式细胞仪上向染料负载细胞悬液中加入浓度为50微摩尔的离奥霉素来制备Indo-1阳性对照。此外，使用抗体偶联荧光团为其他细胞群的单染色质控品准备孔。然后每百万个细胞加入两微克FC受体阻断抗体，并将板孵育45分钟，每15分钟敲击一次以重悬细胞。孵育后，混合并从平板中除去整个体积的细胞，并将其添加到1.7毫升微量离心管中。离心并倒出上清液。用一毫升预热的cRPMI洗涤细胞后，再次沉淀并倒出上清液。将细胞重悬于一毫升cRPMI中并孵育每个试管，使试管顶部略微打开以进行气体交换，从而使细胞内AM酯完全脱酯。接下来，制备所有用户定义的荧光染料偶联抗体的预混液，并将其添加到静息细胞中，以获得一毫升的细胞体积。休息后，沉淀细胞并通过重悬于一毫升冷却至四摄氏度的cRPMI中来洗涤沉淀。重复此步骤并将试管放在冰上。使用带盖的 5 毫升聚苯乙烯流管将单个样品重悬于 500 微升无酚红 cRPMI 中。为了使用钙通量分析保持温度，通过在小水浴中加入浴珠并将其加热至37摄氏度来准备珠浴。小心地将 50 毫升的编年史管切成两半，然后丢弃管的顶部。用浴珠填充剩余管的四分之三，并在珠浴中使其达到 37 摄氏度。将珠浴放在流式细胞仪附近，并用泡沫塑料架放置试管。在流式细胞仪上分析之前，将试管分别加热至37摄氏度7分钟。登录流式细胞仪软件，然后单击库选项卡。然后选择荧光标签以添加钙结合的 Indo-1 和无钙的 Indo-1。然后在荧光标签组下选择紫外激光，然后单击添加以添加荧光标签名称。选择激光激发和发射波长，然后单击保存。继续实验设置。单击采集选项卡，打开/创建新实验并将所需的荧光标签添加到实验中。为实验创建一个参考组和一个新的样本组，并标记这两个组。在“采集”选项卡下，将要记录的事件设置为最大值为 1000 万，停止体积设置为最大体积 3000 微升，停止时间设置为最大 36， 000 秒。采集多色组合中包含的用户定义偶联抗体和 Indo-1 功能染料的单染色对照。向钙结合的Indo-1阳性对照中加入50微摩尔离奥霉素并涡旋。将流管添加到样品进样板，然后单击记录。然后将无钙Indo-1阴性对照添加到流式细胞仪中，然后单击记录。通过单击“取消混合”按钮来解混合单个染色的对照。解混完成后，使用未混合工作表创建连续图，例如 SSC-A 与 FSC-A，用于去除样品中的碎片，SSC-A 与 SSC-H 用于去除双峰。然后通过单击“绘图”创建门。将图添加到工作表中，然后在门内双击以创建下游门控种群。继续，直到考虑所有感兴趣的人口。接下来，将先前制备的单染色对照样品加热至37摄氏度以进行顺序分析。设置流式细胞术软件，在冰水中运行去离子两到三分钟，以确保流式细胞仪的流体稳定性。通过使用面矩形或椭圆门按钮创建门以包括感兴趣的人口，在未混合工作表上设置顺序图。在浇口内双击，将 y 轴和 x 轴参数更改为 SSC-A 而不是 SSC-H。通过左键单击轴完成轴参数的定义。创建具有SSC-A与活性染料信号的图。对胺染料染色呈阴性的活细胞进行门控门。活死染色阴性的活细胞将在y轴和x轴上的零标记处聚集。双击内部图以创建一个仅包含单细胞活淋巴细胞的新图。在 x 轴上将 Y 轴更改为 Indo-1 与时间的关系，以可视化钙内流。接下来，将加热的生物样品放入机器的SIT中。以中等流速每秒 2， 500 至 3000 个事件运行样品，以可视化有限数量的细胞群中的钙内流。在采集控制中将下一个管子添加到珠浴中。按记录以记录30秒的初始数据，以获得样品中钙信号传导的基础水平。然后单击停止并从样品进样口取出试管。将 30 μg 非偶联抗 CD3 直接加入样品管中以引发钙内流。快速将试管放回样品进样口，然后在软件中按记录。记录数据七分钟，观察软件中采集控制下经过的时间。然后按 停止 在软件中。每毫升离奥霉素加入一微克并记录30秒，以获得感兴趣细胞中的最大钙信号。继续下一个样品并重复工作流程，直到收集完所有样品。保存实验并单击导出 zip 文件。未混合的文件被上传到外部流式细胞术分析软件。时间图与侧向散射图中的稳定一致信号表明流体稳定性。对淋巴细胞群进行门控，然后在侧散射高度与面积的图上区分单胞胎。评估单体的生存能力。然后对CD19阴性群体应用门控。这些T细胞亚群使用CD4和CD8抗体通过可视化钙结合的Indo-1荧光与时间的关系来评估。在单个细胞群中，钙结合的Indo-1与时间的关系图显示了刺激前的基线。随着细胞内钙水平下降和添加离子霉素引起的钙反应，达到峰值反应。在添加抗CD3抗体之前，检测到微弱的钙结合Indo-1信号。在峰值响应期间，细胞显示出强烈的钙结合Indo-1信号和减少的无钙Indo-1。在峰后五分钟，Indo-1荧光几乎恢复到基线。加入离奥霉素后，观察到钙结合的Indo-1的强信号，表明细胞的染料负载充分。对稀有种群的分析是通过在每个感兴趣的种群进行门控后绘制钙结合的Indo-1与无钙的Indo-1来进行的。并分析每个子集在整个时间过程中的钙反应。使用全谱分析的钙通量可以在各种免疫亚群中进行高参数流式细胞术分析。用于 T 细胞亚群无偏聚类的高维分析可与高参数流式细胞术结合使用以进行高级分析。该技术是为分析大量样品中多个免疫亚群而开发的。

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

流式细胞仪用来评估染色质的T细胞国家

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

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