A single-cell suspension was prepared for staining infiltrating cells in BALF and MLNs from HDM-sensitized mice. Red blood cells in the specimens were lysed in RBC lysis buffer (BioLegend). The Fc receptor on the prepared cells was blocked with anti-CD16/32 antibody. The cells were then counterstained. Staining for B220, CD3, CD4, and CD8α was conducted to separate B cell and T cell subsets. Staining for B220, CD11b, CD11c, and anti-mouse IgE antibodies was conducted to separate IgE-positive B cells. Staining for CD11b, CD11c, Ly6G, and Siglec-F was conducted to separate neutrophils and eosinophils. Staining for CD3, CD4, CD8α, CD25, CD44, CXCR5, and PD1 was conducted to separate Tfh cells. To stain STING, the cells were treated with fixation/permeabilization buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min at 4 °C after counterstaining with each cell marker. The cells were washed twice with 1 × Perm/Wash buffer (BD Biosciences) and incubated in 2 µg/mL anti-STING antibody in 1 × Perm/Wash buffer for 30 min at 4 °C. Next, the cells were incubated with 0.2 µg/mL mouse anti-rat IgM for 30 min at 4 °C. They were then suspended in staining buffer (1 × PBS (−) containing 0.1% sodium azide and 10% fetal calf serum (FCS)). To stain IL-4, MLN cells from HDM-sensitized mice were cultured 4 h in RPMI1640 complete medium with 100 ng/mL phorbol myristate acetate (PMA, FUJIFILM Wako pure chemical co.), 1 µg/mL ionomycin (FUJIFILM Wako pure chemical co.) and 10 µg/mL brefeldin A (BD biosciences). After the incubation, MLN cells were stained with CD3, CD4, CD8α, CD25, CD44, CXCR5, and PD1 abs for the analysis of Tfh cells. After counterstaining, the cells were fixed with Fixation/Permeabilization buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min at 4 °C. The cells were then washed twice with 1 × Perm/Wash buffer (BD Biosciences) and incubated in 2 µg/mL of anti-IL-4 for 30 min at 4 °C. Finally, the cells were washed twice with 1 × Perm/Wash buffer and suspended in a staining buffer (10% FCS, 0.1% Sodium azide in 1 × PBS) for flow cytometric analysis by FACS Lyric (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (Ashland, OR, USA).

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