J774 mouse macrophages

THP-1 human monocyte cells, Jurkat T cells (ATCC, Manassas, VA, USA) were cultured in RPMI media (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (pen/strep). J774 mouse macrophages (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s MEM (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% FBS, 1% pen/strep. Normal human lung fibroblasts (NHLFs) were obtained from Lonza, Walkersville, MD, USA and cultured in Fibroblast basal Medium-2 (Lonza, Walkersville, MD, USA) with supplements (FGM-2 bullet kit, Lonza, Walkersville, MD, USA). The NHLFs were passaged and used till passage 4. Human Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy volunteers from New York Blood Center upon obtaining informed consent and after Institutional Review Board (IRB) approval. were isolated by Ficoll separation and were maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA) prior to the experiment. All the cells were incubated in 5% CO₂ supply at 37 °C.

J774 mouse macrophages (ATCC, Rockville Pike, MD, USA) were cultured at +37°C in 5% CO₂ atmosphere in Dulbecco´s Modified Eagle´s Medium supplemented with glutamax-1 (DMEM; Invitrogen, Paisley, UK) containing 10% (v/v) heat-inactivated FBS (fetal bovine serum), 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B (all from Gibco, Wien, Austria). For experiments, cells (2.5 x 10⁵ cells/well) were seeded on 24-well plates and the cell monolayers were grown for 72 h before the experiments were started.
Salbutamol, terbutaline and rolipram were dissolved in dimethyl sulfoxide (DMSO), and 8-Br-cAMP and LPS in phosphate buffered saline (PBS). LPS (10 ng/ml) and/or the compounds of interest at the concentrations indicated or the solvent (DMSO, 0.1% v/v) were added to the cells in fresh culture medium containing 10% FBS and the supplements. Cells were further incubated for the time indicated.
The effect of LPS and the tested chemicals on cell viability was evaluated by modified XTT test (Cell Proliferation Kit II; Roche Diagnostics, Mannheim, Germany). Neither LPS nor the other chemicals used in the experiments were observed to evoke cytotoxicity.