Rgm csf

BM neutrophils (0.25 × 10⁶) from WT, Stat4^–/–, and Stat4^fl/flLysM^cre mice were suspended in 0.1 mL RPMI 1640 supplemented with 1% FBS and 10 mM HEPES (“migration media” hereafter) and seeded onto the top well of the Transwell. The bottom wells were loaded with 0.6 mL migration media supplemented with the following: media alone, 12–100 ng/mL rmCXCL1, or 50 ng/mL rGM-CSF (both Peprotech). The loaded cells were allowed to migrate for 3 hours at 37°C before being harvested and counted. The migration index indicates the percentage of transmigrated neutrophils normalized to the mean of the controls for each genotype.
Isolated human peripheral blood neutrophils were resuspended in migration media and incubated for 30 minutes with IL-12 (40 ng/mL) before loading into Transwell inserts (3.0 μm pore, Corning). The top wells of the Transwell chambers were loaded with 0.5 × 10⁶ neutrophils in 200 μL of migration media. The bottom wells were loaded with 0.6 mL migration media either alone or supplemented with 100 ng/mL rhCXCL1 or 50 ng/mL rGM-CSF (both Peprotech). Neutrophils were allowed to migrate for 90 minutes at 37°C before being harvested and counted. The migration index indicates the percentage of transmigrated neutrophils normalized to the mean of the controls for each condition.

Thioglycolate-elicited MΦs were isolated on day 4 from the peritoneum of mice injected intraperitoneally with 0.5 ml of 3% thioglycolate. Resident peritoneal MΦs were recovered from naive mice. BM-MΦs and BM-DCs were generated by culturing bone marrow cells with 20 ng ml⁻¹ rM-CSF (PeproTech) for 7 days and with 16.67 ng ml⁻¹ rGM-CSF (PeproTech) plus rIL-4 (5 ng ml⁻¹, PeproTech) for 6 days, respectively. Cells were stimulated with TLR4-grade LPS from Escherichia Coli O111:B4 (Alexis Biochemicals). To enhance surface CD14 expression, BM-DCs were stimulated with 0.5 μM GpG (ODN 1826, Alexis Biochemicals) for 18 h. Splenic DCs were isolated by CD11c MACs beads (Miltenyi Biotec) and Marilyn cells were purified with CD4⁺ T cells isolation kit II (Miltenyi Biotec).

MLACs and MDSCs were cultured in 10% FBS-RPMI 1640 containing 25% LLC TTCM with 10 ng/ml rGM-CSF (Peprotech). Cells were collected after 5 days of culture, and percentages of F4/80⁺ TAM were analyzed by flow cytometry.