Department of Immunology, Allergology, Rheumatology and the Infla-Med Research Consortium of Excellence, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium.
Department of Immunology and Allergology, AZ Jan Palfijn Gent, Ghent, Belgium.
Cytometry B Clin Cytom. 2020 Nov;98(6):483-490. doi: 10.1002/cyto.b.21868. Epub 2020 Feb 3.
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.
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.
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.
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.
荧光亲和素对外周嗜碱性粒细胞颗粒基质的染色可能是监测嗜碱性粒细胞脱颗粒的可靠技术。本研究比较了基于亲和素的技术与 CD203c 的上调和 CD63 的出现对各种刺激的反应。
研究了 14 名对抗 IgE 有反应的个体、9 名健康对照者和 5 名桦树花粉过敏患者和 5 名无反应者。激活实验包括抗 IgE、fMLP、白细胞介素 (IL)-3 和桦树花粉过敏原。使用抗 CD63、抗 CD203c 和亲和素通过流式细胞术和显微镜分析嗜碱性粒细胞的激活/脱颗粒。
抗 IgE、fMLP 和相关过敏原的刺激导致 CD203c 的上调、CD63 的出现和亲和素结合的增加。对抗 IgE 和过敏原的反应中，CD203c 的上调在 10 分钟内达到峰值，CD63 和亲和素结合在 10-20 分钟后达到平台。CD63 染色导致双峰分布，亲和素染色导致单峰偏移，脱颗粒和非脱颗粒细胞之间的区分不太明显。对 fMLP 的反应中，CD203c 和 CD63 的上调以及亲和素结合在 2.5 分钟后达到最大值。在用抗 IgE 和 fMLP 孵育后，CD203c+细胞的百分比高于 CD63+和亲和素+细胞的百分比，表明激活和脱颗粒之间存在分离。CD63+细胞的百分比系统地高于亲和素+细胞的百分比。仅用 IL-3 孵育会上调 CD203c，而不会观察到 CD63 或亲和素结合。
亲和素对外周蛋白聚糖的染色是定量嗜碱性粒细胞脱颗粒的可靠技术，但与使用 CD63 作为读出的传统检测方法相比，没有提供额外的价值。
