Monocyte nucleofector kit

Neutrophils in BALF and bone marrow (BM) from mice were isolated using Neutrophil Isolation Kit (Miltenyi Biotec) according to manufacturer’s guideline. To obtain BALF, mice were sacrificed by over dosage avertin. The trachea was exposed using scissors. Using a 23 G needle to slowly inject 1 ml cold PBS with 0.1 mM EDTA into the lung, then collect the BALF from lungs. To obtain BM, femurs and shins were isolated with scissors from sacrificed mice, and then the bits of bone were removed. Flush the femurs and shins with 3 ml cold RPMI-1640 medium using a 25 G needle. The BM neutrophils were cultured in RPMI-1640 medium supplemented with 10% FBS and 1000 U/ml penicillin and 1000 μg/ml streptomycin. For plasmid transfection, the expression vector was transfected into BM neutrophils using the monocyte nucleofector kit (Lonza) according to the manufacturer’s instructions. Delivery of HMGB1-siRNAs (RIBOBIO) or PAFR-siRNAs (Santa Cruz) into cells was carried out with Lipofectamine RNA interference MAX Transfection Reagent (Life Technologies) following the manufacturer’s instructions.

THP-1 cells were transfected with the pre-miR-223 (10 μM; Life Technologies), or with a fluorescently-labeled negative control (5 μM) using Amaxa I and the Monocyte Nucleofector Kit (Lonza). Transfected THP-1 cells (~95% based on fluorescence labelling) were stimulated (1–3 h, 37 °C) with BaP (5 μM) or vehicle, followed by LPS (1–8 h, 37 °C). Cytokine levels were determined in supernatants using specific ELISA kits (BioLegend). Concentration was divided by total protein level in cell extracts to correct for cell number in each condition.