Recombinant gm csf

Mouse. Splenic PMN-MDSCs or M-MDSCs isolated from TB mice were treated with mouse IFNβ (2000 units/ml; PBL Assay Science) for 2 h before assessing their suppressive activity. Total BM cells were treated with TES (30% v/v) or thapsigargin (THG) (0.5, 1, 2 μM; Sigma) and recombinant GM-CSF (10 ng/ml) for 16–18 h followed by measurement of cell surface IFNAR1 levels by flow cytometry. BM PMNs treated with lactic acid (20 μM; Sigma Aldrich) and recombinant GM-CSF (10 ng/ml) for 16–18 h followed by flow cytometry analysis. BM PMNs and Mon pretreated with LY2228820 p38 inhibitor (1 μM; Selleckchem) or vehicle (DMSO) for 2 h and then treated with TES (30% v/v) for an additional 2 h. Experiments with hypoxia (0.5% O₂) for 16–18 were maintained using a hypoxic chamber (BioSpherix).
Human. HD PMNs were isolated as described above and treated with TES (30% v/v), TCM (30% v/v) or THG (1 μM; Sigma), and recombinant GM-CSF (20 ng/ml; PeproTech) for 16–18 h. PMNs were pretreated with human IFNβ (2000 units/ml; PBL Assay Science) for 2 h followed by THG (1 μM) for another 16–18 h before suppression assay as described previously.

Bone marrow-dendritic cells (BMDCs) were isolated from mouse bone marrow cells as previously described (Jin et al., 2019a (link)). Briefly, bone marrow cells were isolated and cultured in RPMI 1640 medium containing 20 ng/mL recombinant GM-CSF (Sigma–Aldrich), 20 ng/mL IL-4 (Sigma–Aldrich) and 10% FBS at 37°C and 5% CO₂. Immature DCs were collected on day 7 for further experiments. The DCs were treated with rTs-Serpin (10 μg/mL) alone or combination of rTs-Serpin (10 μg/mL) and β-glucan (50 μg/mL) in vitro for 24 h. Dendritic cells were treated with sterile PBS as a control. Cytokines (IL-12p70 and IL-10) levels in the supernatant were quantified by ELISA (R&D Systems). The stimulated DCs were stained with a FITC-conjugated monoclonal antibody (mAb) to CD11c, APC-conjugated mAbs to CD86 (Biolegend, United States) and PE-conjugated mAbs to MHC-II (Biolegend, United States). The cells were analyzed by using a BD FACSCalibur Flow Cytometer and FlowJo software (Tree star Inc, Ashland, OR) (Jin et al., 2020a ).

Murine bone marrow-derived macrophages (BMDM) were isolated from the bone marrow cells from mouse femurs. Then, the cell suspensions were filtered through a 100-micron nylon cell strainer, collected by centrifugation at 300g for 10 min, and resuspended in DMEM containing 10% FBS, 1% penicillin/streptomycin and GM-CSF (40 ng/mL, Thermo Fisher Scientific, Waltham, MA, USA). BMDMs were cultured at 37 °C with 5% CO₂ for 7 days before all experiments. Recombinant LPS (100 ng/mL, Sigma, St Louis, USA) and recombinant GM-CSF (40 ng/mL) were used in preparing macrophage M1 inducing medium. IL-4 (10 ng/mL, Thermo Fisher Scientific) and recombinant GM-CSF (40 ng/mL) was used in preparing macrophage M2 inducing medium.