Phorbol 12 13 dibutyrate pdb

ROS production was measured using L-012-enhanced chemiluminescence, as previously described (To et al., 2017 (link)). Inflammatory cells isolated from the BAL were seeded into a 96-well OptiView plate (5 × 10⁴ cells/well) with Dulbecco’s Modified Eagle’s Medium (DMEM; Thermofisher, United States) containing 4.5 g/L of glucose, 110 mg of sodium pyruvate and 10% Fetal Bovine Serum (FBS; Sigma-Aldrich, United States), and allowed to adhere for 3 h prior to starting the assay. Cells were then washed with warm 37°C Krebs-HEPES buffer and exposed to a Krebs-HEPES buffer containing L-012 (10⁻⁴ mol/L) (WAKO Chemicals) in the absence (i.e., basal ROS production) or presence (stimulated ROS production) of the protein kinase C (PKC) and NADPH oxidase activator, phorbol 12,13-dibutyrate (PDB; 10⁻⁶ mol/L) (Sigma-Aldrich, United States). The same treatments were performed in blank wells (i.e. with no cells), which served as controls for background luminescence. All treatment groups were performed in triplicate. Photon emission [relative light units (RLU)/s] was detected using the BMGlabtech microplate reader (CLARIOstar, Germany) and recorded from each well for 1 s over 60 cycles. Individual data points for each group were derived from the average values of the three replicates minus the respective blank controls.

Aldara 5% (Meda®, Solnam, Sweden) was stored at room temperature, and weighed to 25 mg when required. Imiquimod (Invivogen, Thermo Fisher Scientific, Carlsbad, USA, cat. no. tlrl-imq) was dissolved in PBS at a concentration of 1 mM and aliquoted into tubes containing 0.5 mL. L-012 (WAKO chemical, Richmond, USA, cat. no. 120-04891) and phorbol 12,13-dibutyrate (PDB; Sigma Aldrich, St Louis, USA, cat. no. P1269-5MG) were dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich, St Louis, USA, cat. no. 472301; 100%) in 10 μL and 5 μL aliquots, respectively at a concentration of 10⁻²M. Chemicals were stored at −20 °C and quickly thawed when required.

LPS (Invivogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and 2-DG (Sigma-Aldrich, St. Louis, MO, USA, Cat no. D8375) were dissolved in H₂O at a concentration of 1 mg/mL and 1 M, respectively. 6-aminonicotinamide (6-AN; Sigma-Aldrich, St. Louis, MO, USA, Cat no. A68203) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA, cat. no. 472301; 100%) at a concentration of 200 mM. L-012 (WAKO Chemicals, Richmond, MO, USA, cat. no. 120-04891) and phorbol 12,13-dibutyrate (PDB; Sigma-Aldrich, St Louis, MO, USA, cat. no. P1269-5MG) were dissolved in DMSO in 10 μL and 5 μL aliquots, respectively, at a concentration of 1 × 10⁻² M. IFN antibody receptor 1 (IFNAR; Bio X cell, Lebanon, NH, USA, Cat no BE0241) was diluted 1:1000 in cell culture medium. Apocynin (Sigma-Aldrich, St. Louis, MO, USA, Cat no. A10809) was dissolved in DMSO at a concentration of 300 mM. Chemicals were stored at −20 °C and rapidly thawed when required, apart from 6-AN and Apocynin, which were freshly prepared before each use.