Apc anti mouse il 4

Basophil test and IL-4 production detection followed a method previously described by Torrero et al. with minor modification [22 (link)]. CD200R was used as a marker of basophil activation and IL-4 was used to evaluate basophil function. 250 μL heparinized whole blood was collected from each mouse. Two samples within group were pooled and then 1 : 1 diluted with DMEM medium. 500 uL diluted blood samples were incubated with or without 1 μg/mL CryJ2 protein at 37°C. One hour later, cells were treated with protein transport inhibitor Golgi-Plug (BD Biosciences, San Jose, CA) and then continued to be cultured for 100 minutes. Cells were stained with IgE-FITC, CD4-PerCP, B220-PerCP, and CD200R-PE (BioLegend, San Diego, CA) for 30 minutes and treated with RBC lysis/fixation buffer (BioLegend). Cells were then intracellularly stained with anti-mouse IL-4-APC (BioLegend) for 30 minutes and analyzed by flow cytometry (BD Accuri C6). Basophils were defined as IgE⁺CD4⁻B220⁻ cells. For basophil activation assays, cut-off gates for CD200R-PE and IL-4-APC positivity were established using the fluorescence-minus-one (FMO) technique.

CD4⁺ T
cells were stimulated using a cell activation cocktail with Brefeldin
A (Biolegend, Beijing, China) for 4 h, incubated with an antimouse
CD4 surface marker (Biolegend, Beijing, China), and then fixed using
a Cyto-Fast Fix/Perm Buffer (Biolegend, Beijing, China) to destroy
the cell membrane. Following this, CD4⁺ T cells were subjected
to staining with antimouse IL-4 APC (Biolegend, Beijing, China). The
sample was subsequently analyzed using flow cytometry, and the resulting
data were imported into FlowJo v10.8.1 software (BD Life Sciences)
for further analysis.