Anti human crth2 percp cy5

The biological analysis was conducted on fresh peripheral blood mononuclear cells (PBMCs) derived from blood samples. To assess the subsets of ILC1, ILC2, and ILC3 in peripheral blood among patients with elevated uric acid (UA), a flow cytometry assay was carried out. Briefly, 1 × 10⁶ cells were suspended in 100 µL phosphate-buffered saline (PBS) prior to antibody incubation. The cells were then incubated with Zombie Aqua fixable viability dye (BioLegend, San Diego, CA, USA), anti-human-CD45-APC/Fire 750 (BioLegend), anti-human-c-Kit-PE/Cy7, anti-human-CRTH2-PerCP/Cy5.5, anti-human-Lineage cocktail (CD3/14/16/19/20/56)-FITC, and anti-human-CD127-BV421 (all from BioLegend) at room temperature for 30 min in the absence of light. Following a wash with 2 mL PBS, the cells were resuspended in 500 µL PBS and examined using flow cytometry (FACS Aria™ II, BD Bioscience, NJ, China). List mode data files were analyzed using FlowJo™ 10 software. Total lymphocytes were identified based on forward and side scatter properties. The innate lymphoid cell (ILC) gate was defined by CD45 + Lineage-CD127 + expression. ILC subtypes were further distinguished as ILC1s (c-Kit-CRTH2-), ILC3s (c-Kit-CRTH2 +), and ILC2s (c-Kit +).

We aimed to confirm that purified αS1‐casein was bound to cell surface sIgE and activated the PS‐basophils. Purified αS1‐casein was labeled with biotin using the Biotin Labeling Kit‐NH2 (Dojindo Molecular Technologies, Kumamoto, Japan). Biotin‐labeled αS1‐casein was added to PS‐basophil suspension and incubated on ice for 30 minutes. After washing, anti‐human IgE‐PE (BioLegend, San Diego, CA), CRA1 (anti‐human FcεRIα‐PE/Cy7, detecting non‐IgE‐binding site on FcεRIα; BioLegend), CRA2 (anti‐human FcεRIα‐FITC, detecting IgE‐binding site on FcεRIα; BioAcademia, Osaka, Japan), anti‐human CD3‐APC/Alexa Fluor 750 (Beckman Coulter), anti‐human CRTH2‐PerCP/Cy5.5 (BioLegend), and streptavidin‐Brilliant violet 421 (BioLegend) were added. The reaction was allowed to proceed for 30 minutes in the dark at 4°C and was stopped by adding the stop solution. After adding Fix and Lyse solution and washing, cells were resuspended in 0.5 mL PBS containing 0.1% formaldehyde and analyzed using the flow cytometer.