Pe cy7 anti il 4

Splenocytes or lymph node (LN) cells were harvested and stained with eFluor780-fixable viability dye (eBioscience), Pacific Blue-anti-CD3e (BioLegend, San Diego, CA, USA), PE-cyanine (Cy)7-anti-CD8 (BioLegend), and PerCP-Cy5.5-anti-CD4 (BioLegend) antibodies. After fixation and permeabilization, the cells were stained with PE-anti-IL-13, PE-anti-IL-22, allophycocyanin-anti-IL-5, PE-Cy7-anti-IL-4, Alexa Fluor 488-anti-IL-17A, and BV785-anti-IFN-γ antibodies (BioLegend). The data were analyzed using FlowJo (Tree Star, Ashland, OR, USA).

Splenocytes were isolated from mice as previously described^{32 (link)}. Briefly, RBCs were treated with lysis solution for 15 min at room temperature, after which 1 × 10⁶ cells were collected for analysis. For T cell activation, cells were incubated at 37 °C in RPMI1640 medium (GIBCO, Grand Island, NY, USA) supplemented with a cell activation cocktail containing brefeldin A (423303; Biolegend, San Diego, CA, USA) according to the manufacturer’s protocol. The activated cells were washed in phosphate-buffered saline (PBS) and incubated in 5% BSA/PBS for 15 min on ice. Immunostaining was then performed using anti-CD4 Alexa Fluor 488-conjugated antibodies (1:500; 100425; Biolegend). After 3 washes with ice-cold staining buffer, cells were resuspended in fixation buffer (420801; Biolegend), incubated for 15 min on ice, and washed. After permeabilization, cells were immunostained with PE/Cy7 anti-IFN-r (1:500; 505825; Biolegend) and PE/Cy7 anti-IL-4 (1:500; 504117; Biolegend) antibodies. Splenocyte staining was analyzed on a Guava EasyCyte mini instrument and data were analyzed using Cytosoft software version 4.2.1 (Merck Millipore, Billerica, MA, USA).

Splenocytes (5 × 10⁶/mL) were stimulated with plate-bound anti-CD3 antibodies (2 μg/mL) and soluble anti-CD28 antibodies (2 μg/mL) for 3 days, and brefeldin-A (5 μg/mL: Biolegend) for the final 16 h of culture. Cells were resuspended in flow buffer (PBS with 0.1% sodium azide and 1% FBS), and stained with anti-CD16/32 TruStain fcX (1: 200, Biolegend) and a live/dead viability dye (BV-510 at 1:500; BD Biosciences) for 30 min at 4 °C. Staining of surface markers (APC-CD4, FITC-CD8α, PercpCy5.5-CD19, all Biolegend) was performed for 30 min at 4 °C in the dark. Cells were fixed, permeabilized using the FIX/PERM set (Biolegend) and blocked in 5% rat serum for 10 min at room temperature prior to intracellular staining for IL-4 with PE-Cy7-anti-IL-4 (Biolegend) for 20 min at room temperature. Data was analyzed using BD FACSDIVA software (BD Biosciences, San Jose, CA, USA).