Recombinant murine gm csf

Bone marrow was collected from tibias and femurs of 4- to 6-month-old female C57BL/6 mice and cultured for 7 days at 37°C and 5% CO₂ in DMEM supplemented with 10% FBS, antibiotics, and 20 ng/mL recombinant murine GM-CSF (BioLegend, 576304). Cells were then washed with PBS to remove nonadherent cells, and adherent BMMs were detached by incubation in TrypLE Select at 37°C (Gibco, Thermo Fisher Scientific, 12563011) followed by gentle cell scraping. BMMs were plated into 96-well plates at 1 × 10⁵ cells/well in growth media lacking GM-CSF and allowed to adhere overnight before use.

Peritoneal cells were from naïve Balb/c mice and seeded at 2 × 10⁶ cells/mL complete medium, allowed to adhere in culture dishes for 1 h in an incubator. Adherent cells were peritoneal macrophages assessed as CD11b⁺F4/80⁺cells by flow cytometer. Splenocytes were from spleen and bone marrow cells were obtained by flushing tibiae and femora of naïve Balb/c mice. Bone marrow–derived macrophages (BMDMs) were obtained by culture of bone marrow cells in a BMDM medium consisting of RPMI 1640, 10% FBS, 1% penicillin/streptomycin, 0.01% 2-β mercaptoethanol (Sigma-Aldrich), and 20 ng/mL of recombinant murine GM-CSF (Biolegend). After 4 days, BMDMs medium was replaced, and adherent cells were harvested after 8 days as BMDM. MH-S and THP-1 wild-type (WT), both from ATCC, were used as alveolar (mouse) and monocyte-like (human) macrophages. THP-1 cells knockdown in NLRP3, ASC and caspase1 were from Invivogen. Cells were cultured in RPMI 1640 complete medium. Hygromycin B selective antibiotic (Sigma-Aldrich) was used at 100 μg/mL for knockdown THP-1 cells. Lipopolysacharride (LPS) from Escherichia coli 0111:B4 (Sigma-Aldrich) was used at 100 ng/mL in some experiments. Phorbol 12-myristate 13-acetate (PMA) from Sigma-Aldrich, was used at 20 ng/mL to activate THP-1 cells; about 1 h before experiments.