Flow cytometric basophil activation tests: Staining of exteriorized basophil granule matrix by fluorescent avidin versus appearance of CD63
Flow cytometric basophil activation tests: Staining of exteriorized basophil granule matrix by fluorescent avidin versus appearance of CD63
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DOI：
10.1002/cyto.b.21868
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发表时间：
2020-02-03
影响因子：
3.4
通讯作者：
Sabato, Vito
中科院分区：
文献类型：
--
作者：
Ebo, Didier G.;Elst, Jessy;Sabato, Vito
研究背景体外嗜碱性粒细胞颗粒基质荧光抗生物素蛋白染色可能是监测嗜碱性粒细胞脱颗粒的可靠方法。本研究比较了基于抗生物素蛋白的技术与上调的CD 203 c和外观的CD 63在响应各种stimulation.Methods 14个人响应抗IgE，9个健康对照，和5个桦树花粉过敏患者，和5个nonresponses进行了研究。激活实验包括抗IgE、fMLP、白细胞介素（IL）-3和桦树花粉过敏原。嗜碱性粒细胞活化/脱粒进行了分析，通过流式细胞术和显微镜使用抗-CD 63，抗-CD 203 c，和avidin.Results刺激抗IgE，fMLP，和相关的过敏原的结果上调CD 203 c，CD 63的外观，并增加抗生物素蛋白结合。在响应抗IgE和过敏原，上调的CD 203 c在10分钟内达到峰值，CD 63和抗生物素蛋白结合10-20分钟后达到一个平台。CD 63染色导致双峰分布，抗生物素蛋白染色导致单峰转变与脱粒和非脱粒细胞之间的不太清楚的歧视。在响应fMLP，上调CD 203 c和CD 63和抗生物素蛋白结合是最大的2.5分钟后。孵育抗IgE和fMLP后，CD 203 c+细胞的百分比高于CD 63+和抗生物素蛋白+细胞，指向激活和脱粒之间的解离。CD 63+细胞的增殖率全身性高于抗生物素蛋白+细胞。孵育IL-3只upregulates CD 203 c，而没有CD 63或抗生物素蛋白binding observed.Conclusions染色的exteriorized蛋白多糖的抗生物素蛋白是一种可靠的技术，以量化嗜碱性粒细胞脱粒，但提供没有附加值相比，传统的检测方法，使用CD 63作为读出。
Background Staining of exteriorized basophil granule matrix by fluorescent avidin might be a reliable technique to monitor basophil degranulation. This study compares the avidin-based technique with the upregulation of CD203c and appearance of CD63 in response to various stimuli.Methods Fourteen individuals responsive to anti-IgE, nine healthy controls, and five birch pollen-allergic patients, and five nonresponders were studied. Activation experiments included anti-IgE, fMLP, interleukin-(IL)-3, and birch pollen allergen. Basophil activation/degranulation was analyzed by flow cytometry and microscopy using anti-CD63, anti-CD203c, and avidin.Results Stimulation with anti-IgE, fMLP, and relevant allergen results in upregulation of CD203c, CD63 appearance, and an increase in avidin binding. In response to anti-IgE and allergen, upregulation of CD203c peaks within 10 min, CD63 and avidin binding reach a plateau after 10-20 min. CD63 staining leads to a bimodal distribution, avidin staining causes a unimodal shift with a less clear discrimination between degranulating and nondegranulating cells. In response to fMLP, upregulation of CD203c and CD63 and avidin binding are maximal after 2.5 min. Following incubation with anti-IgE and fMLP, percentages of CD203c+ cells are higher than those of CD63+ and avidin+ cells, pointing to a dissociation between activation and degranulation. Percentages of CD63+ cells are systemically higher than those of avidin+ cells. Incubated with IL-3 only upregulates CD203c, while no CD63 or avidin binding is observed.Conclusions Staining of exteriorized proteoglycans by avidin is a reliable technique to quantify basophil degranulation but offers no added value when compared to traditional assays that use CD63 as a readout.