Dharmafect reagent 3

Mouse BM from femurs and tibias was resuspended in HBSS-EDTA, and BMDMs were isolated, cultured, and activated for 5 d as previously described (Mosser and Zhang, 2008 (link)). For NLRP3 inflammasome activation, 10⁶ cells/ml BMDM were treated with LPS (100 ng/ml) for 3 h followed by ATP (1 mM; InvivoGen) for 3 h or vehicle controls. For neutrophil isolation, BM was resuspended in HBSS-EDTA and layered on a Percoll gradient (52, 69, and 78%) in 1× HBSS. Neutrophils were removed from the 69/72% interphase. Cells were washed twice with HBSS and seeded at 10⁶ cells/ml in OptiMEM supplemented with 1 µg/ml aprotinin. NLRP3 was activated by 3-h LPS priming (100 ng/ml) followed by 3-h stimulation with 10 µM nigericin. After the combined treatments, cell-free supernatants were harvested, and cells were washed and harvested for either mRNA or protein extraction and analysis.
For functional studies assessing the role of miR-223 in the Nlrp3 binding site–deficient mice, primary monocytes were isolated using anti-CD14 magnetic beads from WT or nlrp3^m223del BM (Miltenyi Biotec) and differentiated by incubation with 100 ng/ml M-CSF for 3 d. Primary monocytes were transfected with 25 nM control scrambled or miR-223 mimic using Dharmafect reagent 3 (Thermo Fisher Scientific) and activated with 1 µg/ml LPS and 5 mM ATP (Sigma-Aldrich) as previously described (Haneklaus et al., 2012 (link)).