Amr 100

The effect of CGE on the cell viability was determined using MTT assay. 3T3-L1 preadipocyte was seeded in a 96-well plate with 1 × 10⁴ cells/well of inoculum density, and different concentrations (0.0~4.0 mg/mL) of CGE were treated. After 48 h of cultivation, 0.1 mL MTT solution was added to induce formation of formazan at 37 °C and 0.1 mL DMSO was added after 4 h to dissolve formazan. Then, absorption was measured at 540 nm using a microplate reader (AMR-100, Allsheng, Seoul, Korea). In accordance with international standards (ISO 10993-5; Part 5: tests for in vitro cytotoxicity), the cytotoxic level was set below 85% cell viability [30 ]. The cell viability was calculated with the following equation: $$\mathrm{Cell}\mathrm{viability}\left(\%\right)=\left\{1-\frac{\mathrm{Abs}\left(\mathrm{sample}\right)}{\mathrm{Abs}\left(\mathrm{control}\right)}\right\}\times 100$$
Sample: absorbance with CGE addition, control: absorbance with DMEM addition.

BLs extracted from P. acidilactici K10 (K10 BL) or P. acidilactici HW01 (HW01 BL) (20, 40, or 80 μg/mL) were added to wells of a 96-well culture plate and pre-incubated for 3 h at 37 °C. L. monocytogenes (1 × 10⁷ CFU/mL) was then added to wells of the culture plate and incubated for an additional 24 h. After incubation, planktonic L. monocytogenes was removed, and L. monocytogenes biofilm was gently washed with PBS. The biofilm was stained with 0.1% crystal violet for 30 min at room temperature and washed with PBS twice to remove excess stain. The adherent stain was dissolved with 0.1% acetic acid and 95% ethanol, after which the biofilm was quantified by measuring the OD at 595 nm using a microplate reader (AMR-100, Allsheng, Hangzhou, China).

The reducing power of brassinin was measured with a slight modification of a previously published method [31 (link)]. The FRAP working solution was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine, 20 mM ferric chloride, and distilled water in a ratio of 10:1:1:1.2. A total of 6 μL of brassinin and 200 μL of FRAP reagent were added to a 96-well plate and incubated at 37 °C for 4 min. Then, absorbance was measured at 520 nm with a microplate reader (AMR-100, Allsheng, Hangzhou, China). Ferric sulfate was measured at the same concentration as brassinin, and a standard curve was constructed using the average FRAP value versus concentration. This was used to calculate the antioxidant capacity of brassinin.

HGF-1 cells were seeded in a 35 mm confocal dish (SPL) at a concentration of 2 × 10⁴ cells/mL and incubated for 24 h. After incubation, cells were washed with phosphate-buffered solution (PBS, Gibco), treated with 1 mL of prepared composite eluates, and incubated for another 24 h. The negative control (NC) was treated with DMEM and the positive control (PC) was treated with 1 mM H₂O₂ (Sigma-Aldrich).
For live/dead assay, a Live/dead Viability kit (Invitrogen, Waltham, MA, USA) was used and observed under an inverted fluorescence microscope (DS-Ri2, Nikon Corporation, Tokyo, Japan) and a confocal laser microscope (LSM 700, Carl Zeiss, Thornwood, NY, USA). Live cells were observed with green fluorescence, and dead cells were observed with bright red fluorescence.
For WST-1 assay, the EZ-Cytox cell viability assay kit (DoGen Bio, Seoul, Korea) was used to determine cytotoxicity after 24 h of treatment. The optical density (OD) was determined at 450 nm using a microplate reader (AMR-100, Allsheng, Hangzhou, Zhejiang, China). Relative cell viability was calculated as the ratio of OD of experimental groups (TN, CX, and DN) to that of NC. The experiments were triplicated (n = 9).

GIST48B (5,000 cells/well), GIST54 (10,000 cells/well), and GIST226 (15,000 cells/well) were plated in a 96-well flat-bottomed plate (Grenier, Germany) and cultured in IMDM for 24 h before treatment with bortezomib. Viability studies were performed at 3 and 6 days after bortezomib treatment in KIT-independent GIST cell lines. 200 μL MTT (5 mg/ml) for each well was added into GIST cells and incubated for 4 h in the dark. Finally A₄₉₀ was measured by an automatic enzyme-linked immunosorbent assay system AMR-100 (Hangzhou Allsheng Instruments Co.,Ltd., Zhejiang, China). The data were normalized to the DMSO control group. All experimental points were set up in quadruplicate wells and repeated three independent experiments.