Recombinant murine il 4

Murine dissociated neurons were prepared as previously described (Walsh et al., 2015 (link)). After 5 d in vitro, neurons were treated with 50 ng/ml murine recombinant IL-4 (Peprotech) or PBS for 7 d. Cells were homogenized in lysis buffer, and RNA was isolated using RNeasy micro kit (Qiagen) according to the manufacturer’s instructions. RT-qPCR was performed as previously described (Jolivel et al., 2013 (link)). Primers are listed in Table 2. Ribosomal protein S29 (RPS29) was used as a housekeeping gene. For RT-qPCR on tissue, mice were perfused with PBS and their brains were removed for dissection of the cortex and hippocampus. Spleens were removed shortly before perfusion. Tissue was processed to RNA and qPCR for IL-4 (Table 2) and RPS29 was performed.

All cell lines used were routinely grown in DMEM (Invitrogen, UK) supplemented with 10% fetal bovine serum (Labtech, International, UK) and 1% l-glutamine (PAA, UK). Equal numbers of BV2 microglia or RAW 264.7 macrophages were seeded onto 12- or 96-well plates (Greiner Bio-one, CellStar). Cells were treated with 100 ng/ml LPS (E. coli derived, 0111:B4) (Sigma-Aldrich, UK) or 20 ng/ml murine recombinant IL-4 (Peprotech, UK) in DMEM for polarization to the pro- or anti-inflammatory phenotypes, respectively, or left untreated for the unpolarized control phenotype. At the end of the experiment, cells were fixed in 4% PFA (in PBS, pH 7.2) for immunofluorescence staining.
4T1-GFP, BV2, and RAW 264.7 cells were seeded onto 96-well plates and when cell confluency reached 60–70%, cells were treated with mannosylated control (PBS) or clodronate (10 mg/ml) liposomes (Encapsula Nanosciences, USA) diluted 1:50 or 1:100 in culture medium for 24 h. Alternatively, BV2 and RAW 264.7 cells seeded onto 96-well plates were treated with IL-4 (20 ng/ml) or LPS (100 ng/ml) for polarization when cell confluency reached 40–50%. 24 h after treatment with the polarizing agents, cells were treated with mannosylated control or clodronate liposomes diluted 1:50 in culture medium for 24 h. At the end of the experiment cells were subjected to the MTT viability assay.

Bleomycin (BLM) was obtained from Huirui. Murine recombinant IL‐4 was purchased from PeproTech. The Lipofectamine 3000 transfection kit was acquired from Invitrogen. Cholesterol and DSPC were purchased from Sigma‐Aldrich, Inc. Lipidoid (C12‐200) was acquired from Xinjiahecheng Medical Chemistry Corporation. mPEG2000‐DEG was purchased from NOF Corporation. siRNAs targeting Mecp2 and Scr siRNA were purchased from Guangzhou RiboBio Co., Ltd.
Antibodies against CD68, CD206, and TGF‐β1 were purchased from Santa Cruz Biotechnology. Antibodies against arginase‐1 and fibronectin were purchased from Abcam. Antibodies against Mecp2, IRF4, inducible nitric oxide synthase (iNOS), and α‐SMA were purchased from Cell Signaling Technology. Antibodies against collagen I, Irf4, Gapdh, and β‐actin were purchased from Proteintech, and antibodies against Ym1 were purchased from Thermo Fisher Scientific. APC‐conjugated anti‐mouse F4/80, PE‐conjugated anti‐mouse CD11c, and FITC‐conjugated anti‐mouse CD206 antibodies were purchased from BioLegend.

Trypticase soy broth was procured from Fisher Scientific (Hampton, NH, USA). Fetal bovine serum (FBS) and gentamicin (catalog no. 15750060) were purchased from Gibco-BRL (Grand Island, NY, USA). α-Minimal essential medium (α-MEM), anti-α-actin (A2228) antibody, rosiglitazone, polyinosinic-poly(C) [poly(I·C)], and lysostaphin (L7386) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Macrophage colony-stimulating factor (M-CSF), in the form of CMG 14-12 supernatant, and glutathione S-transferase RANKL (GST-RANKL) were prepared as previously described (33 (link)). Human M-CSF and Ficoll histopaque were purchased from Invitrogen (Carlsbad, CA, USA). Human CD14 magnetic beads were obtained from Miltenyi Biotec (Auburn, CA, USA). Anti-NFATc1 (7AG) antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-histone H3 (96C10) was obtained from Cell Signaling Technology (Beverly, MA, USA). Murine recombinant IL-4 and IFN-γ were purchased from Peprotech (Rocky Hill, NJ, USA).

Bone marrow-derived murine DC (BMDC) were generated by standard methods [27 (link)] with the following modifications: Bone marrow cells were obtained from C57BL/6 mice and washed in RPMI 1640 media. The cells were then placed in non-tissue culture treated T75 flasks at a concentration of 1 x 10⁶ cells per ml in 20 ml complete RPMI (RPMI 1640 with 10% FBS, 20 μg/ml gentamycin sulfate, 50 μM 2-mercaptoethanol) containing 20 ng/ml murine recombinant GM-CSF and 10 ng/ml murine recombinant IL-4 (Peprotech, Rocky Hill, NJ)). Cells were cultured at 37°C, 5% CO2. On day 3, media was replaced with fresh complete RPMI containing cytokines. On day 5, cells were harvested, washed and resuspended in complete RPMI at 5 x 10⁵ cells/ml.

Bone marrow from wild-type C57BL/6, OX40L^fl/fl, CD11c^cre OX40L^fl/fl, or OX40L^+/Hu-CD4 reporter mice were isolated by flushing femurs and tibias with RPMI, supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 1% L-glutamine and 1% sodium pyruvate (cRPMI). Bone marrow cells were strained through a 70 μm filter, centrifuged, and resuspended in RBC lysis buffer for 2 min on ice. Cells were plated in cRPMI in tissue-culture-treated 10 cm dishes. 20 ng/mL murine recombinant GM-CSF (Peprotech) and 5 ng/mL murine recombinant IL-4 (Peprotech) was added to generate bone-marrow derived dendritic cells (BMDC). Half of the medium was removed on day 2 of differentiation and new pre-warmed medium supplemented with GM-CSF and IL-4 (2X concentrations) were added. The culture medium was entirely replaced on day 3 with fresh warmed cRPMI + GM-CSF (20 ng/mL) only. On day 6, non-adherent cells in the culture supernatant were harvested for tumour cell-line co-culture.