Anti mouse il 4 antibody

The following antibodies were used for the present study.
Anti-mouse CD3, clone number 17A2, BioLegend, 05112-40-100, V450;
Anti-mouse CD4, clone number GK1.5, BioLegend, 100434, PerCP/Cyanine5.5;
Anti-mouse CD8, clone number 53-6.7, BioLegend, 100714, APC/Cyanine7;
Anti-mouse CD25, clone number 3C7, BioLegend, 101908, FITC;
Anti-mouse CD69, clone number H1.2F3, BioLegend, 104508, PE;
Anti-mouse CTLA-4, clone number UC10-489, BioLegend, 106305, PE;
Anti-mouse PD-1, clone number 29F.1A12, BioLegend, 135210, APC;
Anti-mouse CD19, clone number 1D3, eBioscience, 12-0193-82, PE;
Anti-mouse CD45, clone number 30F11, BioLegend, 103122, Alexa Fluor 488;
Anti-human CD45, clone number HI30, BioLegend, 304025, PerCP;
Anti-mouse IFNγ, clone number XMG1.2, eBioscience, PE;
Anti-mouse IL-17A, clone number TC11-18H10.1, BioLegend, Alexa Fluor 647;
Anti-mouse IL-13, clone number 13A, BioLegend, PE/Cyanine7;
Anti-mouse Foxp3, clone number MF14, BioLegend, PE;
Anti-mouse TNFα, clone number MP6-XT22, BioLegend, Alexa Fluor 488;
Purified Anti-mouse IFNγ antibody, clone number XMG1.2;
Purified anti-mouse IL-4 antibody, clone number 11B11.

Spleens from wild type or PEG C57Bl/6 mice were excised and mashed with a 40 μm mesh nylon strainer, washed with PBS, and resuspended at a concentration of 1*10⁸ cells/mL in PBS supplemented with 2% FBS (VWR Seradigm cat #1500-500) and 1 mM EDTA (Sigma-Aldrich, cat #E7889). Naïve T cells were isolated using Stemcell^TM EasySep^TM Mouse Naïve CD4⁺ T Cell Isolation Kit (Cat# 19765) and re-suspended at a concentration of 1*10⁶ cells/mL in complete RPMI media in a 24 well plate with 25μL/mL Dynabeads® Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific Cat# 11456D), 20 ng/mL recombinant human IL-12 (Biolegend Cat# 577002) and 5 µg /mL anti-mouse IL-4 antibody (Biolegend Cat# 504108). After 48 h of incubation at 37 °C, Dynabeads® were removed from T cells using Stemcell^TM EasySep^TM Magnet (Cat# 18000). At day 2 cells were counted and resuspend at 0.2 × 10⁶ cells/mL in complete RPMI and 20 ng/mL IL-12, 5 µg/mL anti-mouse IL-4, and 25 ng/mL IL-2 (Peprotech Cat# 200-02). After 48 h of incubation cells were resuspended in complete RPMI supplemented with 25 ng/mL IL-2 at 0.5 × 10⁶ cells/mL. Day 7 expanded cells were used for all experiments. Notably, we observe no differences in phenotype, proliferative capacity and effector T cell function using mouse vs human recombinant IL-2.