Immunomagnetic negative selection kit

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.

Spleens were isolated from healthy mice or mice after two weeks of EAE induction with or without BBR3378 treatment and homogenized between two frosted glass slides in PBS with 2% fetal bovine serum (FBS). Tissue debris was then sedimented, supernatant containing splenocytes collected, red blood cells removed by using a lysis buffer (Thermo Fisher Scientific, Waltham, MA), and monocytes isolated by using an immunomagnetic negative selection kit (STEMCELL, Vancouver, Canada). Monocytes (2x10⁶ cells per 60 mm dish) were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37° C in a 5% CO2 enriched environment for five days and subjected to analysis of macrophage markers.
To obtain macrophages that exhibit a robust response to lipopolysaccharides (LPS), primary monocytes from healthy animals were collected and cultured as described previously with addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) (PromoCell, Heidelberg, Germany) for seven days to induce macrophage differentiation, and then activation of these MDMs by Escherichia coli 026:B6 LPS (Thermo Fisher Scientific, Waltham, MA) was assessed.