Ly2228820 p38 inhibitor

Mouse. Hematopoietic progenitor cells (HPCs) were isolated from mouse bone marrow by using a Lineage depletion kit (Miltenyi), according to the manufacturer’s instructions. Cells were seeded at 25,000 cell/ml in 24-well plates and recombinant GM-CSF (20 ng/ml; Invitrogen), 20% v/v TES were added on day 1 and day 3. At day 5, Ly6G positive neutrophils were isolated by using anti-Ly6G biotin (Miltenyi) and streptavidin beads (Miltenyi), according to manufacturer’s followed by suppression assay. In addition, total cells were stained and analyzed for flow cytometry. In other experiments, LY2228820 p38 inhibitor (1 μM; Selleckchem) was added to HPC culture on day 3.
Human. Hematopoietic progenitor cells (HPCs) were isolated from cord blood using the CD34 MicroBead Kit (Miltenyi), according to the manufacturer’s instructions. Cells were seeded at 5 × 10⁴ cell/ml in 6-well plates with recombinant G-CSF (100 ng/ml; PeproTech) and GM-CSF (10 ng/ml; PeproTech). At day 7, 30% v/v TES were added and the next day flow cytometry analysis was performed.

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.