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首页> 外文期刊>Cytometry, Part A: the journal of the International Society for Analytical Cytology >Coregistered Spectral Optical Coherence Tomography and Two-Photon Microscopy for Multimodal Near-Instantaneous Deep-Tissue Imaging (Apr, 10.1002/cyto.a.24012, 2020)
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Coregistered Spectral Optical Coherence Tomography and Two-Photon Microscopy for Multimodal Near-Instantaneous Deep-Tissue Imaging (Apr, 10.1002/cyto.a.24012, 2020)

机译：用于多式联近瞬时深层组织成像的内心光谱光学相干断层扫描和二光子显微镜（APR，10.1002 / CYTO.A.24012,2020）

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The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow-cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real-time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo-3 (Ca2+), ION NaTRIUM Green-2 (Na+), and ION Potassium Green-2 (K+). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR-1 agonist), AYPGKF (PAR-4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. (c) 2020 International Society for Advancement of Cytometry
机译：血小板激动剂与它们各自的膜受体的相互作用触发细胞内信号传导，其中细胞溶离子通量在活化过程中起重要作用。虽然接受细胞间自由钙的关键贡献，但在最近的研究中，血小板活化中的钠和钾的作用较小。在这里，我们实施了一种新的流式细胞术方法，用于在血小板活化时监测时间细胞溶质的游离钙，钠和钾离子通量，我们证明了离子动力学的实时可视化的可行性，特别是浓缩钠和钾。用选择性离子指示剂，Fluo-3（Ca2 +），离子Natrium Green-2（Na +）和离子钾绿色-2（K +）加载血小板。通过流式细胞术监测荧光。在测量稳定的基线后，激活血小板，随着时间的推移获得离子指示剂荧光，最多10分钟。用血栓素类似物U46619，ADP，凝血酶，TRAP6（PAR-1激动剂），AYPGKF（PAR-4激动剂），癌症（胶原受体GPVI激动剂）或其组合用血小板激活。我们评估了预型参数（特别是染料加载时间和浓度）以实现准确的方法。随后，我们以血小板激动剂为特征细胞溶胶钙，钠和钾动力学。我们观察了不同的激动剂协同作用模式。总之，目前的工作突出了通过流式细胞术的使用细胞溶质离子监测，以研究血小板活化后的特征钙，钠和钾长型。这种易于技术开启了一种新的方法来分析不同血小板亚群中的信号传导，并且应该证明对于调查血小板病理生理学。 （c）2020国际促进细胞计中的国际社会

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