Balb/c 3T3-A31 fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM – D5648 Sigma–Aldrich), CHIR-99021 cost supplemented with 10% fetal bovine serum (FBS-GIBCO) at 37 ± 1 °C, 90 ± 10% humidity, 5.0 ± 1.0% CO2/air. The cells were removed from the culture flasks using trypsinization (trypsin:EDTA solution at a 0.25%:0.02% ratio) when they exceeded 50% confluence but prior to reaching 80% confluence. Cell viability was evaluated using the Trypan blue exclusion method with a Neubauer chamber. A cell suspension containing 3 × 104 cells/mL was prepared on the day of plate seeding using culture medium supplemented with 10% FBS. The peripheral wells (blanks) of the 96-well microtiter plates

were seeded with 100 μL of routine culture medium and the remaining wells received 100 μL of a suspension containing 3 × 104 cells/mL (3 × 103 cells/well). The plates were incubated for 24 ± 2 h (37 ± 1 °C; 90 ± 10% humidity, 5.0 ± 1.0% CO2/air) to allow the cells to form a monolayer of less than 50% confluence. This incubation period assured cell recovery, adherence and progression to the exponential growth phase. Each plate was examined under a phase contrast microscope to identify experimental and systemic cell seeding errors. For in vitro assays, the terpenes (nerolidol, α-terpineol, L(−)-carvone, (+)-limonene, L-menthone, DL-menthol, PD0325901 order pulegone or 1,8-cineole) were prepared individually as

a micellar suspension to allow dissolution in water. The micelles were prepared as follows: 10 mg of phosphatidylcholine (PC) and 50 μL of the terpenes to be tested were dissolved in 50 μL of ethanol. The mixture was sonicated for 10 min in a Ti-probe sonicator to obtain a homogeneous dispersion of small micelles. The micellar suspension was prepared without terpenes for control groups. The experimental samples were directly diluted Hydroxychloroquine in culture medium (DMEM) to obtain the concentration of use and filtered through a syringe-filter with a PES TPP® membrane (0.22 μm pore size)

to assure sterility. The final concentration of ethanol in all cultures was lower than 0.05%. A Balb/c 3T3-A31 cell suspension containing 3 × 104 cells/well was seeded in 96-well plates, and after a 24 h recovery period, the plates were treated with eight different concentrations of freshly prepared test compounds in complete medium (six wells per concentration) and incubated for an additional 48 h. The control wells (blanks) received complete culture medium supplemented with 10% FBS. Subsequently, 250 μL of neutral red (NR) medium was added to all wells, including the blanks, and incubated (37 ± 1 °C, 90 ± 10% humidity, 5.0 ± 1.0% CO2/air) for 3.0 ± 0.1 h. The cells were briefly observed 2–3 h after incubation for NR crystal formation. After 3 h, the NR medium was removed and the cells were carefully rinsed with 250 μL/well of pre-warmed PBS.