Il 13 pe

For cytokine staining, cells were restimulated with 20 nM PMA and 1μM ionomycin for 4 hours, and 5μg/ml brefeldin A was added during the last 2 hours of restimulation. Cells were stained with the viability dye eFluor780 (eBiosciences; Cat # 65-0865-14), and then fixed in 4% paraformaldehyde for 8 minutes at room temperature. Cytokine staining was done in permeabilization buffer containing 0.05% saponin. For transcription factor staining, unstimulated cells were fixed, permeabilized, and stained using the FoxP3 Staining Kit (eBiosciences; Cat # 00-5523-00). Samples were analyzed with a flow cytometer (BD LSR II). Mouse antibodies: IL-13-PE (eBiosciences; eBio13A), IL-4-APC (eBiosciences; 11B11), IL-5-PE (Biolegend; TRFK5), IFNg-FITC (eBiosciences; XMG1.2), TNFa-AlexaFluor700 (BD Biosciences; MP6-XT22), and GATA-3-PE (eBiosciences; E50-2440). Human antibodies: IL-13-FITC (eBiosciences; PVM13-1), IL-4-APC (BD Biosciences; 8D4-8), IL-17A-eFluor450 (eBiosciences; eBio17B7), and IL-17F-PE (BD Pharmingen; 079-289).

To examine effector cytokine production, single-cell suspension of SVF was stimulated with following concentrations of PMA and Ionomycin (Sigma-Aldrich) in the presence of 1 μg/ml brefeldin A (BD Bioscience) for 4 hours at 37°C in a CO₂ incubator. For IFN-γ production from T cells and NK cells, PMA (10 ng/ml) and Ionomycin (1μg/ml) were used. For IL-5, IL-13 production from ILC2, and IL4 from eosinophils, PMA (40ng/ml) and Ionomycin (500ng/ml) were used. Cells were then washed, surface stained, fixed, and permeabilized using fix perm buffer (eBioscience), followed by intracellular staining with IFN-γ-APC, IL-5-APC, IL-13-PE, and IL4–FITC antibodies from eBioscience.

The following antibodies were used for surface staining (all from BioLegend): CD117 (Kit)-APC/Cy7 (or APC); FcεRIα-PE; Ly6G (Gr1)-PE/Cy7 (or fluorescein isothiocyanate [FITC]); and CD11b (Mac1)-Pacific blue. Intracellular cytokine staining was performed exactly as described (Deho' et al., 2014 (link); Rusca et al., 2012 (link)). Briefly, cells were stimulated with 1 μg/ml IgE-anti-DNP and 0.2 μg/ml HSA-DNP (both from Sigma) for 3 hr, with the addition of 10 μg/ml brefeldin A in the last 2 hr of stimulation. Cells were fixed with 4% paraformaldehyde prior to permeabilization and staining with the following antibodies: IL-6-PE, TNF-α-PE/Cy7 (BioLegend), and IL-13-PE (eBioscience).

Single lung or LN cell suspensions (1×10⁶ cells/100 μl) were incubated in FACS buffer (PBS, 1% FBS, 0.05% NaN₃) containing CD16/32 Fc-blocking antibody (2.4G2, BD Biosciences, San Jose, CA) for 30 minutes at 4°C and then stained with fluorescently-labeled antibodies for 30 minutes at 4°C. Cells were washed repeatedly in FACS buffer, fixed in 2% PFA for 20 minutes at room temperature, washed and resuspended in FACS buffer for flow cytometry analysis. Data was acquired using the LSR II flow cytometer (BD Biosciences) and analyzed using FACSDiVa (BD Biosciences) and FlowJo software (TreeStar, Inc. Ashland, OR). The following mouse-specific monoclonal antibodies were purchased from eBioscience with the clone indicated in parentheses: CD11c-Alexa Fluor 647 (HL3), CD11b-PE-Cy7 (M1/70), Gr1-APC-eFluor780 (RB6-8C5), CD317-Alexa Fluor 488 (eBio927), CD80-PE (16-10A1), CD86-PE (GL1), PD-L1-PE (MIH5), B7-DC-PE (TY25), CD4-PE-Cy7 (RM4-5), IL-13-PE (eBio13A) and IL-17A-Alexa Fluor 647 (eBio17b7). Myeloid DCs were defined as CD11c⁺CD11b⁺Gr1⁻CD317⁻.