Recombinant mouse il 4

Bone marrow derived dendritic cells (BMDCs) were generated using Iscoves Modified Dulbecco’s medium (IMDM) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin streptomycin solution, recombinant mouse granulocytes macrophage colony stimulating factor (GM-CSF, 20 ng/ml, Peprotech), and recombinant mouse IL-4 (10 ng ml⁻¹, Thermo Fischer Scientific). Non-adherent BMDCs at day 6 culture were collected and plated (0.5 × 10⁶ cells /well) in 24 well plates. Cells were treated with PKM2 allosteric agonist ML265 (100 μM), 2-DG (10 mM), anti-ST2/IL-33R (2 μg ml⁻¹) or control (DMSO) for two hours before Alternaria (2 μg ml⁻¹) treatment. Culture supernatants were collected after 24 hours for ELISA. For knockdown of PKM2, primary bone marrow derived cell line (NR-9456) were obtained from BEI Resources, NIAID, NIH and maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% irradiated fetal bovine serum. Briefly, cells were transfected with either 1 nM anti-PKM2 siRNA or 1 nM scrambled siRNA (Dharmacon) using DharmaFECT. Silencing efficiency were determined after 72 hours by qRT-PCR and Western blot using anti-PKM2 and anti-histone 3 (H3) antibody (Cell Signaling Technology).

Murine B cells cultured at a concentration of 5 × 10⁵ cells/ml in RPMI medium supplemented with 15% FBS, penicillin-streptomycin (100 units/ml) and L-glutamine (2 mM) were treated with anti-CD40 antibody (1 μg/ml, eBioscience) and recombinant mouse IL-4 (20 ng/ml; PeproTech). Cells were collected at the indicated time points. Class switch recombination (CSR) was measured by staining with PE-labeled anti-mouse IgG1 (BD Biosciences) and APC-labeled anti-mouse CD45R/B220 (BD Biosciences). The expression of CD40 and FAS was measured by staining with APC-labeled anti-mouse CD40 (BioLegend) and APC-labeled anti-mouse CD95 (BioLegend), respectively. Data acquisition was performed using a FACSVerse flow cytometer (BD Biosciences) (Cheong et al., 2016 (link)).

On day −1, NB-21.2D9 cells were seeded into 96-well plates at 1,000 cells/well in B-cell media (BCM); RPMI-1640 medium (Invitrogen) supplemented with 10% HyClone FBS (Thermo scientific), 5.5 × 10⁻⁵ M 2-mercaptoethanol, 10 mM HEPES, 1 mM sodiumpyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1 × MEM nonessential amino acid (all Invitrogen). Next day (day 0), recombinant mouse IL-4 (Peprotech; final 2 ng/ml) was added to the cultures, and then single B cells were directly sorted into each well using cell sorters [FACSVantage or FACSAria (both BD Biosciences)]. On day 2, 50% (vol.) of the culture media were removed from cultures and 100% (vol.) of fresh BCM were added to the cultures. On days 3 to 8, two-thirds of the culture media were replaced with fresh BCM every day. On day 9, culture supernatants were harvested for ELISA determinations and culture plates were stored at −80°C for V(D)J amplifications.