Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix.
Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix.
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DOI：
10.3389/fcell.2018.00074
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发表时间：
2018
影响因子：
5.5
通讯作者：
Furuno T
中科院分区：
文献类型：
--
作者：
Yokawa S;Suzuki T;Hayashi A;Inouye S;Inoh Y;Furuno T
脱颗粒是指炎症介质的分泌，如组胺、血清素和蛋白酶，它们储存在肥大细胞的颗粒中，引发过敏反应。这些释放介质的量已经用细胞质量进行了生物化学测量。为了研究活单细胞的脱粒过程，传统上使用荧光显微镜观察颗粒的消失和这些颗粒在细胞膜内通过膜融合排出的外观以及颗粒在细胞内的运动。在这里，我们开发了一种视频速率生物发光成像方法，通过测量高斯荧光素酶（GLase）与其底物coelenterazine之间的酶促反应产生的发光活性，直接检测单个肥大细胞的脱颗粒。据报道，神经肽Y （NPY）在分泌颗粒中与血清素共定位，融合到GLase （NPY-GLase）中，在大鼠嗜碱性白血病（RBL-2H3）细胞中，使用人类首选密码子优化基因有效表达。对表达NPY-GLase的RBL-2H3细胞进行生物发光成像分析，结果表明，静息细胞的发光信号可以忽略不计，而在添加抗原后，可反复检测分泌的NPY-GLase的发光信号。此外，该成像方法适用于观察粘附在细胞外基质（ECM）上的RBL-2H3细胞的脱粒情况。这些结果表明，使用glass的视频速率生物发光成像将是检测粘附在多种ECM蛋白上的单个肥大细胞脱颗粒的有用工具。
Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.