S2gpu05re

Mouse bone marrow cells were harvested from the femur and tibia with 8 seconds centrifugation at 11,000xg in the presence of sterile ACK lysing buffer (0.15 M NH₄Cl, 1 mM KHCO₃, and 0.1 mM Na₂EDTA). Bone marrow cells were further enriched for monocytes using an immunomagnetic negative selection kit from STEMCELL Technologies (catalog#19861) as per manufacturer’s instructions. 0.2×10⁶ freshly isolated monocytes per well were cultured in 96-well tissue-culture-treated flat-bottom plates (Corning, catalog#353072) with 0.22 μm filtered (Millipore, catalog#S2GPU05RE) complete medium (RPMI with 10% fetal bovine serum and 1% penicillin/streptomycin) in a water-jacket incubator for 1 hour at 37°C with 5% CO₂. Non-adhered cells were removed by aspiration and cultured with serum-free medium (RPMI with 1% penicillin/streptomycin) with or without treatments for 16 hours at 37°C with 5% CO₂. Conditioned media were harvested for IL-1β release measurements via ELISA (ThermoFisher Scientific, catalog# 88701388). Cells were lysed with RIPA lysis buffer (ThermoFisher Scientific, catalog#89901) with protease and phosphatase inhibitor cocktail (ThermoFisher Scientific, catalog#78430). Total protein concentration was measured with the BCA protein assay (ThermoFisher Scientific, catalog#23227). Measurements were taken from distinct samples.