Recombinant murine il 4

Splenic naïve CD8⁺ T cells were purified by using Naive CD8a⁺ T Cell Isolation Kit, mouse (Miltenyi Biotec) following the manufacturers protocol, and polarized in vitro toward differentiated Tc1 and Tc2. In brief, naive cells were seeded into anti-CD3e (2 μg/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 μg/ml, clone 37.51, eBioscience) coated plates. The medium contained the following cytokines and/or antibodies:
Tc1 subtype: recombinant murine IL2 (10 ng/ml, R&D Systems), recombinant murine IL12 (10 ng/ml, R&D Systems) and neutralizing anti-IL4 (10 μg/ml, clone 11B11, eBioscience). Tc2 subtype: recombinant murine IL2 (10 ng/ml, R&D Systems), recombinant murine IL-4 (10 ng/ml, R&D Systems) and neutralizing anti-IFNg (10 μg/ml, clone XMG1.2, eBioscience).

Total BM cells were flushed with PBS from mouse femurs and tibiae. For BM-derived DC (BMDC) culture, BM precursors were cultured in RPMI-1640 medium supplemented with 10% FBS (vol/vol), 10 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, R&D), and 4 ng/ml recombinant murine IL-4 (R&D). Non-adherent cells were removed and fresh BMDC culture medium with GM-CSF and IL-4 was added at day 3. Mature DCs were harvested for analysis at day 7. For BM-derived macrophage (BMDM) culture, BM cells were cultured in DMEM medium supplemented with 10% FBS (vol/vol) and 15 ng/ml recombinant murine macrophage colony-stimulating factor (M-CSF, R&D). Half of the medium was changed with prewarmed fresh medium with M-SCF after 60 h. Mature macrophages were harvested for analysis at day 5. For neutrophil isolation, BM cells were suspended in 45% percoll (GE Healthcare) and laid on the top of 62% and 81% percoll gradient, and then centrifuged at 1,500 × g for 30 min at room temperature. Mature neutrophils were collected from the interface of 62% and 81% percoll.

Splenic naive T helper cells were purified with the CD4⁺CD62L⁺T Cell Isolation Kit II (Miltenyi Biotec) and polarized in vitro toward differentiated Th2 subtype as described before in [32 (link)]. In brief, naive cells were seeded into anti-CD3e (2 μg/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 μg/ml, clone 37.51, eBioscience) antibody coated 96-well round bottom plates. The medium contained the following cytokines and/or antibodies for Th2 subtype: recombinant murine IL-2 (10 ng/ml, R&D Systems), recombinant murine IL-4 (10 ng/ml, R&D Systems), and neutralizing anti-IFN-g (5 μg/ml, cloneXMG1.2eBioscience). The cells were removed from the activation plate on day 4 (72 h). Th2 cells were cultured for another 2 days in the absence of anti-CD3 and CD28 stimulation. Then, cells were restimulated by anti-CD3e/CD28-coated plate for 6 h. For flow cytometric detection, cells were treated with monensin (2 μM, eBioscience) for the last 3 h.

Mouse BMDCs were generated by culturing bone marrow cells in the presence of 10 ng/ml GM-CSF and 10 ng/ml IL-4 for 6 days, as described previously (31 (link)). Briefly, bone marrow cells from the femur and tibia of naive C57BL/6 mice were harvested under sterile conditions, and 2 × 10⁶ cells were seeded into each well of a 24-well cell culture plate and cultured in complete medium (CM) [RPMI 1640 medium containing 10% fetal calf serum (FCS)] with the addition of 10 ng/ml recombinant murine GM-CSF and 10 ng/ml recombinant murine IL-4 (R&D Systems). The cells were cultured at 37°C in a humidified atmosphere of 5% CO₂. On day 7, the suspended cells and loosely adhered cells were collected for further experiments by gently pipetting the medium against the plate several times. For phenotypic characterization, BMDCs were stained with CD11c, CD11b, MHCII, and F4/80 and analyzed by flow cytometry. Cells with highly expressed CD11c, CD11b, and MHCII as well as merely expressed F4/80 were considered successful induction of BMDCs. For LPS treatment, an initially dose of 50 ng/ml was applied; and another dose of 20 ng/ml was added 48 h later if cells need to be cultured more than 2 days.