Gm csf

Potassium hydroxide (KOH), methanol (MeOH), dichloromethane (CH₂Cl₂), Span 85 (SP85), and isopropanol (C₃H₈O) were purchased from Chengdu Kelong Chemical Engineering Company (Chengdu, China). γ-Cyclodextrin (γ-CD) was purchased from Aladdin (Shanghai, China). RPMI 1640 medium, fetal bovine serum, and penicillin-streptomycin were obtained from Gibco (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibodies were obtained from Abcam Ltd (Hong Kong). GM-CSF and IL-4 were produced from Novoprotein(Suzhou, China). ELISA kits were ordered from Enzyme-Linked Biosystems (Shanghai, China). CCK-8 kit was purchased from BOSTER Biological Technology (USA).
Scanning electron microscope (SEM) was measured with JEOL-JSM-7500. X-Ray powder diffraction (XRD) was collected using Bruker D8 advance. The UV/Vis and fluorescence spectra results were detected using Varioskan LUX. Fourier transform infrared (FTIR) spectra were recorded by American Nicolet 67.

Primary bone marrow-derived DCs isolated from the femur and tibia of C57BL/6 mice were cultured with 10 ng/ml IL-4 and GM-CSF (Novoprotein) at 37°C for 4 days. Immature DCs were then collected and purified with Histodenz (Sigma-Aldrich; Merck KGaA). DC suspensions with a purity >85% with CD11C and MHC-II double-staining were used for subsequent assays. NK cells were positively selected from the spleen (SPL) of C57BL/6 mice using the Mouse CD49b Positive Selection kit (cat. no. 18755, Stemcell Technologies, Inc.). CD8⁺ T cells were negatively isolated from SPL and lymph nodes (LN) from OT-1 mice according to the manufacturer's protocol (cat. no. 19835A; Stemcell Technologies, Inc.). The isolated CD8⁺ T cells pre-labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (eBioscience; Thermo Fisher Scientific, Inc.) were added after DCs co-cultured with NK cells in the presence of R848 for 24 h. Following 3 days of co-culture, IFN-γ⁺ CD8⁺ T and CD8⁺ T proliferation was analyzed by flow cytometry.

(1) Cell lines. ID8 mouse ovarian cancer cells were donated by Professor Dong Chen (Institute of Immunology, Tsinghua University), and B16F10 mouse melanoma cells were purchased from the Cell Bank of the Shanghai Branch of the Chinese Academy of Sciences. Cells were cultured in high-glucose DMEM (Corning) supplemented with 10% FBS (Corning) and 1% penicillin-streptomycin (Haoyang Biological Manufacture). All cells were cultured in a 37°C, 5% CO₂ cell incubator. (2) Activated T cells. We collected WT mouse spleen-derived T cells and labeled with CFSE. Then, these T cells were activated by anti-CD3 and anti-CD28 antibodies (BioLegend). (3) Assessment of the immunosuppressive ability of BM-derived CD11b⁺Ly6G⁺Ly6 C^low cells. WT mice BM-derived CD11b⁺Ly6G⁺Ly6C^low cells with or without 100 ng/mL mouse GM-CSF (Novoprotein, Shanghai, China) and 100 ng/mL mouse IL-6 (Novoprotein) stimulation cocultured with activated T cells for 96 hours. The FITC signal intensity of T cells was detected by FCM and the concentration of IL-2 in the supernatants was determined by a commercial ELISA kit (Proteintech, Wuhan, China). (4) Establishment of the primary PMN-MDSCs culture system in vitro. Primary mouse BM-derived CD11b⁺Ly6G⁺Ly6C^low cells obtained by the method described above were cultured with 100 ng/mL GM-CSF (Novoprotein) and 100 ng/mL IL-6 (Novoprotein) and used in subsequent experiments.