Anti il4 pe cf594

BLN was collected on day 20 from the mice in the four groups: asthma, stress, tolerized, and stress/tolerized groups. The BLN cells were preincubated with anti-CD16/CD32 (FC gamma III/II receptor; BD Biosciences, Franklin Lakes, NJ, USA) to reduce the nonspecific binding of the subsequent antibodies. Dead cells were excluded using the LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). The cells were stained for surface antigens with anti-CD3ε-PerCP-Cy5.5 (clone 145-2C11; Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD4-AF700 (clone GK1.5; BD Biosciences), or isotype control antibodies. For intracellular staining, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich), ionomycin (1,000 ng/mL; Sigma-Aldrich), and monensin (2 μm; BioLegend, San Diego, CA, USA) for 4 h before surface antigen staining. After fixation and permeabilization, the cells were incubated with anti-Foxp3-PE-Cy7 (clone FJK-16s; Thermo Fisher Scientific), anti-IL-17A-APC-Cy7 (clone.TC11-18H10.1; BioLegend), anti-IL4-PE-CF594 (clone 11B11; BioLegend), or an isotype control antibody. We considered CD3⁺CD4⁺Foxp3⁺ cells as Treg cells, CD3⁺CD4⁺IL-4⁺ cells as Th2 cells, and CD3⁺CD4⁺IL-17a⁺ cells as Th17 cells. The cells were counted on a FACSAria II flow cytometer (BD Biosciences), and the analyses were performed using the FACSDiva software (BD Biosciences).