The mouse leukemic macrophage cell line, RAW 264.7 (ATCC—TIB-71), was cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) non-inactivated Fetal Bovine Serum, 100 μg/mL streptomycin and 100 U/mL penicillin. The cell line was maintained at 37 °C, in a humidified atmosphere of 95% air and 5% CO2, and used after reaching 80–90% confluence. Cells were plated at 3 × 105 cells/well and left to stabilize for 12 h.
For cell treatments, essential oils, 1,8-cineol (extra pure; Merck, Darmstadt, Germany) and fenchone (99.9% purity; Fluka AG, Buchs, Switzerland) were initially diluted in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., St. Louis, Mo, USA) at a 1:1 proportion and, then, serial dilutions were performed in culture medium. MG-132 (Z-Leu-Leu-Leu-CHO, Boston Biochem, Cambridge, MA, USA) was also dissolved in DMSO (Sigma-Aldrich Co). In both cases, the final concentration of DMSO did not exceed 0.1% (v/v). LPS from Escherichia coli 026:B6 (Sigma-Aldrich Co.) was dissolved in phosphate buffered saline (PBS). The concentrations of the essential oils, 1,8-cineol, fenchone, MG-132 and LPS, and the experimental treatment periods, are indicated in figures and/or figure legends.
For cell treatments, essential oils, 1,8-cineol (extra pure; Merck, Darmstadt, Germany) and fenchone (99.9% purity; Fluka AG, Buchs, Switzerland) were initially diluted in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., St. Louis, Mo, USA) at a 1:1 proportion and, then, serial dilutions were performed in culture medium. MG-132 (Z-Leu-Leu-Leu-CHO, Boston Biochem, Cambridge, MA, USA) was also dissolved in DMSO (Sigma-Aldrich Co). In both cases, the final concentration of DMSO did not exceed 0.1% (v/v). LPS from Escherichia coli 026:B6 (Sigma-Aldrich Co.) was dissolved in phosphate buffered saline (PBS). The concentrations of the essential oils, 1,8-cineol, fenchone, MG-132 and LPS, and the experimental treatment periods, are indicated in figures and/or figure legends.