Cocktail a

The spleen tissue was aseptically removed and prepared into a single-cell suspension. For Th17 (CD3⁺CD4+IL17A+) cells and Treg cells (CD3⁺CD4⁺ CD25 + FOXP3+) detection, the cells were stimulated by cocktail A for 4 h (BD, Cat# 550583). Samples were then washed and re-suspended in 1 × PBS stained with FVS 780 (BD, Cat#565388) to discriminate viable cells and then incubated with various surface markers. Consequently, samples were fixed by eBioscience Fix/Perm (Cat#00-5523-00) or BD Fix/Perm buffer kit (Cat#554714) to destroy the cell membrane and then were stained with FOXP3 (eBioscience, Cat#17-5773-82), IL-17A (BD, Cat# 564169). In the study, CD3 (BD, Cat# 557,666), CD4 (BD, Cat# 552,775) and CD25 (BD, Cat# 558642) were used. Finally, samples were analyzed with the LSR Fortessa cell analyzer (BD) and BD FACSDiva 8.0.3 software.

Collecting spleen tissue and prepareing it into single cell suspension. Pay attention to the aseptic operation. For flow cytometry, cells were stimulated in complete RPMI containing 2 μl cocktail A (BD, Cat#550583) for 4 Construction of eosinophilic asthma mouse h, after blocked by Fc-receptor blocker (BD, Cat#513141) in 37°C for 40Construction of eosinophilic asthma mouse min, washed, and resuspended in 1 × PBS, and stained with FVS780 (BD, Cat#565388) to discriminate viable cells. The eBioscience Fix/Perm (Cat#00-5523-00) and BD Fix/Perm (Cat#554714) buffer kits were used to fix and permeabilize the cells. The intracellular staining Foxp3 (eBioscience, Cat#17-5773-82), IFN-γ (BD, Cat#557735), IL-4 (BD, Cat# 562915), or IL-17A (BD, Cat#564169) were used. Finally, we analyzed the data by the LSR Fortessa cell analyzer and Diva software (BD).
Multi-cytokine detection containing IL-4, IFN-γ, IL-5, IL-13, TGF-β, IL-6, IL-10, and IL17A was performed in accordance with the premixed AimPlex™ multiplex-assay kits (Cat#T2C0710709 and Cat#B111206). OVA-specific IgE in the serum was detected in accordance with the protocol (Cayman, Cat#500840).