Gm csf

Six to ten-week-old male Balb/c mice were euthanized by overdose of inhalant anesthetic and sacrificed. The femurs and tibias were removed, cleaned, and sterilized. The bone marrow was flushed from bones by use of a syringe containing culture medium. For BMDC isolation, the bone marrow cells were washed and cultured at 4.0×10⁶ cells/dish (10 cm culture dish) in culture medium supplemented with 40 ng/ml GM-CSF (Wako, Japan) for 8 days. Harvested cells were blocked with anti-mouse Fc receptor (BioLegend) and then stained with FITC-labeled anti-CD11c (N418, BioLegend) and PE-labeld anti-mouse MHC-II (I-A/I-E; M5, BioLegend) antibodies. CD11c⁺ MHC-II⁺ cells were sorted by BD FACSAria II (BD Biosciences, CA) and used as BMDC (purity >95%, CD11c⁺ MHC-II⁺ cells). For analysis of the function of LG2055 against BMDC, BMDC (5.0×10⁵ cells/mL) were cultured with or without LG2055 (20 µg/mL) in presence or absence of SB505124 (5 µM, Sigma) in 12 well plate (BD Bioscience) for 48 h. After incubation, culture supernatants were collected for measurement of cytokines and total RNA were prepared from BMDC for real time-PCR analysis.

Three human ovarian cancer cell lines, SKOV3, ES2 and RMG1, were purchased from the American Type Culture Collection (Manassas, VA, USA), and a mouse ovarian cancer cell line (iMOC) was a kind gift from Professor Hideyuki Saya (Keio University, Tokyo, Japan)27 (link). The cells were maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) (Gibco, Grand Island, NY) and regularly tested and found to be negative for Mycoplasma contamination.
Peripheral blood mononuclear cells were acquired from healthy volunteer donors; written informed consent for sample collection and subsequent analysis was obtained from all healthy donors. All protocols using human macrophages were approved by the Kumamoto University Hospital Review Board (No. 486), and conducted in accordance with the approved guidelines. CD14⁺ monocytes were purified from the peripheral blood mononuclear cells via positive selection using magnetic-activated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured with GM-CSF (10 ng/ml, Wako, Tokyo, Japan) or M-CSF (50 ng/ml, WAKO) for seven days to differentiate the cells into macrophages. The differentiated macrophages were then used as human monocyte-derived macrophages (HMDMs) in the present study, as described previously28 (link).

Recombinant mouse cytokines (M-CSF, IFN-γ, IL-4, IL-1β and IL-10) were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human cytokines (GM-CSF, M-CSF, IFN-γ, IL-4, IL-1β and IL-10) were purchased from Wako (Osaka, Japan).

For tumor-infiltrating Treg cell preparation, tumor tissues were harvested 3 weeks after tumor inoculation. Tumor tissues were cut into small pieces, incubated in 5% FBS RPMI-1640 in the presence of an enzyme mixture (Miltenyi Biotec) at 37℃ for 45 min, and digested by using a gentleMACS Dissociator and tumor dissociation kit (Miltenyi Biotec), according to the manufacturer’s instructions. Cells were filtered through 70 μm nylon mesh and subsequently centrifuged using different concentrations of Percoll (Sigma-Aldrich) to exclude tissue debris and were washed with staining medium.
BMDCs were generated as described previously (Nakahashi-Oda et al., 2016 (link)). Briefly, bone marrow cells were cultured in a 10 cm culture dish in complete RPMI-1640 containing 10 % FBS in the presence of 10 ng/ml GM-CSF (WAKO) and 10 ng/ml IL-4 (WAKO) for 7 days. BMDCs were enriched by using CD11c MACS Beads (Miltenyi Biotec) to remove dead cells generated during BMDC development.