Didier G Ebo1,2, Jessy Elst1, Michel van Houdt1, Isabel Pintelon3, Jean-Pierre Timmermans3, Tatsuo Horiuchi4, Margaretha A Faber1, Margo M Hagendorens1,5, Christel M Mertens1, Vito Sabato1,2. 1. Department of Immunology, Allergology, Rheumatology and the Infla-Med Research Consortium of Excellence, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium. 2. Department of Immunology and Allergology, AZ Jan Palfijn Gent, Ghent, Belgium. 3. Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium. 4. Department of Anesthesiology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan. 5. Department of Pediatrics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium.
Abstract
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.
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.
Authors: Theodore Kim; Jing Yu; Henry Li; Mark Scarupa; Richard L Wasserman; Athena Economides; Martha White; Carla Ward; Atul Shah; Douglas Jones; Melinda Rathkopf; Kelly Frye; Ahmet Aybar; Shahrooz Shayegan; Benjamin Enav; Laura Ispas; Denise Loizou; David Fitzhugh; James Tracy; James Friedlander; Zachary Jacobs; Jonathan Matz; David Golden; Donald McNeil; William McCann; Christopher Copenhaver; Jeffrey Factor; Raavi Gupta; Oral Alpan; Matthew Plassmeyer; Søren Ulrik Sønder Journal: Cytometry B Clin Cytom Date: 2021-02-04 Impact factor: 3.248