Cd63 fitc

The immune phenotype of EVs was analyzed using flow cytometry (BD FACS Aria III, BD Bioscience, East Rutherford, NJ, USA) by immunostaining with the CD49e-PE (Sony, Tokyo, Japan), CD63-FITC (Biolegend, San Diego, CA, USA), Sca1-APC/Cy7 (BioLegend), CD45-PE/Cy7 (BioLegend), CD9-APC (Biolegend) and CD44-APC/Cy7 (BioLegend) antibodies.

The basophil activation test
was performed using 100 mL of heparinized whole blood, incubated with
20 μL of stimulation buffer (1 M HEPES buffer containing 0.78%
NaCl (w/v), 0.037% KCl (w/v);, 0.078% CaCl₂ (w/v), 0.033%
MgCl₂ (w/v), 0.1% HSA (w/v), 10 μL/mL of IL-3 (1
mg/mL), and 1 μL of monoclonal antibody CCR3-APC (1 mg/mL) (BioLegend
INC, San Diego, CA) for 10 min at 37 °C. Then, 100 μL of
each purified Sola l 7, Sola l 6, and their digests was added at different
concentrations following a 10-fold dilution pattern (0.1–0.00001
μg/mL) and incubated for 30 min at 37 °C. Anti-human IgE
(0.5 mg/mL) (BD Biosciences, Franklin Lakes, NY) or PBS were used
as a positive and negative controls, respectively. After 5 min on
ice to stop the degranulation process, samples were incubated with
1 mg/mL of monoclonal antibodies anti-CD 203c-PE and CD63 FITC (BioLegend)
for 15–20 min at 4 °C. Finally, lysis solution (BD Biosciences)
was used for red cells disruption. Cells were analyzed using a FACSCalibur
flow cytometer (BD Biosciences), acquiring at least 500 basophils
per sample. Results are presented as the percentage of activated basophils
(CD63⁺CD203c⁺CCR3⁺). Due to the low
availability of patients, only the sera of one tomato allergic patient
sensitized to Sola l 6 and Sola l 7 and displaying anaphylaxis (patient
numbers 13 and 7, respectively) was used for this experiment.

Blood granulocytes and single-cell suspensions obtained from digested biopsies of heathy donors and IBD patients were stained with the following antibodies CD66b PE-Cy7 (#305116 clone G10F5, 1/20 dilution, BioLegend), CD16 PerCP (#302028 clone 3G8, 1/20 dilution, BioLegend), CD62L APC-Cy7 (#304814 clone DREG-56, 1/20 dilution, BioLegend), CD69 BV421 (#310930 clone FN50, 1/20 dilution, BioLegend), CD193 FITC (#310720 clone 5E8, 1/20 dilution, BioLegend), CD63 FITC (#353005 clone H5C6, 1/20 dilution, BioLegend) and CXCR4 PE (# FAB170P clone 12G5, 1/10 dilution, R&D systems) and Zombie Aqua Fixable Viability Kit 1/1000 (#423101, BioLegend) for death cells. Cells were fixed using BD Stabilizing Fixative [BD], acquired using a BD FACSCanto II flow cytometer (BD) and analyzed with FlowJO software (version 10.6.1, BD).