Raw 264.7 murine macrophages

RAW264.7 murine macrophages were purchased from the Korean Cell Line Bank (KCLB^®, Seoul, Korea); rat liver Ac2F cells and human oral epidermoid cancer cells (KB) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured at 37 °C in a 5% CO₂ humidified incubator and maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, Nissui, Tokyo, Japan) containing 100 mg/mL streptomycin, 2.5 mg/L amphotericin B, and 10% heat-inactivated fetal bovine serum (FBS). Suspensions of tested cell lines (cal. 1.0 × 10⁴ cells/well) were seeded in 96-well culture plates, cultured for 12 h, and then treated with various diluted concentrations of (+)-(R,E)-6a1 for 24 h, 48 h, and 72 h, respectively. Control cultures were treated with culture medium alone. The tested compounds were evaluated at twice-fold dilutions, and the highest concentration was 50 μM. Cell viability was evaluated using water soluble tetrazolium (WST) reagent (EZ-CyTox, Daeil Lab Service Co., Ltd., Seoul, Korea), which was added to each well (10 μL) and incubated at 37 °C for 1 h. Absorbances were read using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 450 nm. Cells in the exponential phase were used for all experiments.

RAW264.7 murine macrophages were purchased from the Korean Cell Line Bank (KCLB^®, Seoul, Korea), and rat liver Ac2F cells and human embryonic kidney 293T cells (HEK 293T) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured at 37 °C in a 5% CO₂ humidified incubator and maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM, Nissui, Tokyo, Japan) containing 100 mg/mL streptomycin, 2.5 mg/L amphotericin B and 10% heat-inactivated fetal bovine serum (FBS). Cells were seeded in a 96-well culture plates and cultured for 12 h and then treated with various concentrations of PD1 for 24 h. Cell viabilities were evaluated using water soluble tetrazolium (WST) reagent (EZ-CyTox, Daeil Lab Service Co., Ltd., Seoul, Korea), which was added to each well (10 μL) and incubated at 37 °C for 1 h. Absorbances were read using a iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 450 nm.

RAW264.7 murine macrophages (Korea Cell Line Bank, Korea) were incubated in Dulbeccós modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were cultured at 37°C under 5% CO₂ conditions. RAW 264.7 macrophages (0.9 × 10⁵ cells) were seeded in 24-well plates and incubated for 24 h. Then, the cells were stimulated with 25 or 100 μg/ml of GFB 8102 for 6 h. The culture media was collected and centrifuged to abtain a supernant, which was used to measure the amount of pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6.