Multimodel plate reader

With the aforementioned analysis, Metabolite 1 was the main product in our study. Therefore, we selected it for future work. Murine macrophage cells (RAW264.7), human lung adenocarcinoma cells (A549), fibroblast cells (3T3-L1), and melanoma cells (B16BL6) were cultured in a 5% CO₂ atmosphere at 37°C. Cytotoxicity of ginsenoside Rh2, ginsenoside Rg3, and the new metabolite, Metabolite 1, was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay following our previous procedure. The cell lines were seeded at a density of 1–1.2 × 10⁵ cells/mL in a 96-well plate (NEST®, Nest Scientific USA Inc.,USA) in triplicate. After culturing for 24-h, the cells were treated with Rh2, Rg3, and Metabolite 1 (1, 10, 50, and 100 μM). After treatment was finished, 10 μL of MTT solution (5 mg/mL) was mixed to each well, and the plates were incubated for another 4 h. The media containing MTT reagent was then removed from each well, and 100 μL of dimethyl sulfoxide was added. After completion of this step, the absorbance of the reaction solution was measured at 570 nm/630 nm using a multimodel plate reader (BioTek Instruments, Vermont, USA).

The human cell lines A549 and HT29 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and Korean Cell Line Bank (Seoul, Republic of Korea). RPMI 1640 culture medium (GenDEPOT Inc., Barker, TX, USA), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), was used to grow the cell lines at 37°C in a humidified incubator with a 5% CO₂ atmosphere. Cell viability was determined by MTT assay. For MTT assay, cells were seeded in 96-well plates at a density of 1×10⁵ cells/mL. After 24 hours of incubation, the cells were treated with various concentrations of BSA-Rh2 NPs and standard ginsenoside Rh2 for 24 hours. After incubation, 10 μL of the MTT stock solution (5 mg/mL) was added to each well and incubated for 4 hours. Then, the supernatants were removed and replaced with 100 μL of dimethyl sulfoxide (DMSO). The amount of formazan formed by viable cells was measured using multi-model plate reader (BioTek Instruments, Winooski, VT, USA) at a test wavelength of 570 nm with a reference wavelength of 630 nm. In addition, cytotoxicity assay was conducted in HaCaT skin cell lines to analyze the cell viability.31 (link)

The viability of the cells was assessed by MTT (3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyletrazolium bromide) assay. Briefly, the cells were plated at a concentration of 1×10⁵ in a 96-well plate (NEST^®, New Jessey, USA). After 24 hours, the cells were subjected to the following treatment for 48 hours: D-AgNPs (1 µg/mL, 10 µg/mL, and 100 µg/mL), D-AuNPs (1 µg/mL, 10 µg/mL, and 100 µg/mL), CK (1 µM, 2.5 µM, 5 µM, 10 µM, 25 µM, and 50 µM), D-AuNPs 50 µg/mL + CK (1 µM, 2.5 µM, 5 µM, 10 µM, 25 µM, and 50 µM), and D-AgNPs 50 µg/mL + CK (1 µM, 2.5 µM, 5 µM, 10 µM, 25 µM, and 50 µM). Afterward, 10 µL of MTT (5 mg/mL) was added to each well and incubated for 3 hours. Then, media containing treatment and MTT reagent were removed by aspiration and replaced with 100 µL of Dimethyl Sulfoxide (DMSO). The plates were covered with aluminum foil and kept in an incubator for 30 minutes for dissolution of the formed formazan crystals. The amount of formazan crystals formed by viable cells was measured using a multimodel plate reader (Bio-Tek Instrument, Winooski, Vermont, USA) at a test wavelength of 570 nm and compared to a reference wavelength of 630 nm. The percentage of cytotoxicity was defined as ([absorbance of treated cells]/[absorbance of control cell] ×100). Similarly, the toxic effects of D-AgNPs and D-AuNPs on HaCaT cell viability were measured by MTT assay following the same methods.