全谱流式细胞术分析原代小鼠T细胞受体诱导的钙内流
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 12 well TC treated plates | Cell Treat | 229111 | |
| 50 mL conical | Greiner Bio1 | 41-12-17-03 | 50 mL Polypropylene centrifuge tubes with cap |
| 5mL polysterene flow tubes | Corning | 352052 | |
| 5mL syringe | BD syringe | 309646 | plunger only is used sheith is discarded |
| 70uM filter | Greiner bio1 | 542070 | |
| aCD3 (17A2) | Biolegend | 100202 | |
| AKC lysis Buffer | Gibco | A1049201 | |
| Aurora Spectral Flowcytometer | https://cytekbio.com/pages/aurora | ||
| Bath Beads | coleparmer | Item # UX-06274-52 | |
| CD19 PE | Tonbo | 50-0193-U100 | |
| CD1d Tetramer APC | NIH | ||
| CD25 PECy7 | ebioscience | 15-0251 | |
| CD4 APC Cy7 | Tonbo | 25-0042-U100 | |
| CD8a FITC | ebioscience | 11-0081-85 | |
| Cell Incubator | Formal Scientific | ||
| Dissection Tools forceps | McKesson | #487593 | Tissue Forceps McKesson Adson 4-3/4 Inch Length Office Grade Stainless Steel NonSterile NonLocking Thumb Handle 1 X 2 Teeth |
| Dissection Tools Scissors | McKesson | #970135 | Operating Scissors McKesson Argent™ 4-1/2 Inch Surgical Grade Stainless Steel Finger Ring Handle Straight Sharp Tip / Sharp Tip |
| DPBS 1x | Gibco | 14190-136 | DPBS |
| EGTA | Fisher | NC1280093 | |
| FBS | Hyclond | SH30071.03 | lot AE29165301 |
| FlowJo Software | https://www.flowjo.com/ | ||
| Indo1-AM Ester Dye | ebioscience | 65-085-39 | Calcium Loading Dye |
| ionomycin | Millipore | 407951-1mg | |
| Live/Dead Ghost 540 | Tonbo | 13-0879-T100 | |
| Microcentrifuge tubes 1.7mL | Light Labs | A-7001 | |
| Penicillin/Streptomycin/L-Glutamine | Gibco | 10378-016 | |
| PRN694 | Med Chem Express | Hy-12688 | |
| Purified Anti-Mouse CD16/CD32 (FC Shield) (2.4G2) | Tonbo | 70-0161-M001 | FC Block |
| RPMI | Gibco | 1875093 | + phenol red |
| RPMI phenol free | Gibco | 11835030 | -phenol red |
| Table top centrifuge | Beckman Coulter | Allegra612 | |
| TCRβ PerCP Cy5.5 | ebioscience | 45-5961-82 | |
| TCRγ/δ Pe Cy5 | ebioscience | 15-5961-82 | |
| Vi-Cell Blu Reagent Pack | Product No: C06019 | Includes Tripan | |
| Vi-Cell Blu | Beckman Coulter | ||
| Waterbath | Fisher Brand Dry bath |
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