Elisa plate reader at 540

The murine macrophage cell line RAW 264.7 was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). RAW 264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, streptomycin (100 µg/mL), and penicillin (100 unit/mL) at 37 °C under 5% CO₂ humidified incubator. Exponential phase cells were used throughout the experiments. Then, RAW 264.7 cells (1.5 × 10⁴ cells/mL) plated in 24-well plates were fore 16 h and then treated with LPS (1 µg/mL) plus aliquots of the five marine brown algal methanol extract. The cells were then incubated for an additional 24 h at 37 °C under 5% CO₂ humidified incubator. After incubation, the cytotoxic assessment was performed using an MTT assay. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured using an ELISA plate reader at 540 nm (BioTek Instruments, Inc., Winooski, VT, USA). The optical density of the formazan generated in non-treated control cells was considered to represent 100% viability.

First, cytotoxicity was evaluated before measuring the cell proliferation activity of eight compounds isolated from Chinese chive. The cytotoxic assessment was performed according to the method described in Muthuramalingam et al. (2019) [36 (link)] with a slight modification. Briefly, after 24 h of cell seeding with 1 × 10⁵ cells per well (96 well plate), isolated compounds were treated for 24 h. Afterward, cytotoxic assessment was performed using MTT assay. The formazan crystals were dissolved in DMSO, and the absorbance was measured using an ELISA plate reader at 540 nm (BioTek Instruments, Inc., Winooski, VT, USA). The optical density of the formazan generated in non-treated control cells was considered to represent 100% viability. To assess the skeletal muscle cell proliferation activity, activities were determined using BrdU assay (Millipore, Billerica, MA, USA) according to the method used in Kim et al. (2019) [37 (link)]. Briefly, after 48 h of the initial cell seeding with 5 × 10⁴ cells per well (96 well plate), the growth medium (DMEM containing 10% FBS) was replaced with differentiation medium (DMEM containing 2% HS) and treated phytochemicals every other day during differentiation period. Finally, six days after the differentiation induction, cell proliferation activity was measured using BrdU assay.