Multiskan g0

Drug sensitivity in 2D culture systems was tested by the MTT assay. When cells reached the period of logarithmic phase, they were trypsinized into single cells with a density of 1 × 10⁴ cells per well in 96-well plates (180 μL/well). After 12 h of incubation, the 20 μL medium containing various concentrations drugs (5, 10, 15, and 20 μM for MCF-7; and 10, 20, 30, and 40 μM for SMMC-7721) or a drug-free medium (control) were added. After 48 h of incubation, the media were removed and washed with PBS, replaced by 200 μL of 0.5 mg/mL MTT solution in RPMI 1640 media. Following 4 h of incubation in this condition, the MTT solution was removed and 200 μL DMSO was added, and the plates were shaken for 10 min. The optical density (OD) of each condition was measured using a microplate reader (Multiskan G0, Thermo Scientific, Rockford, IL, USA) at a wavelength of 570 nm with a reference wavelength of 630 nm. Then, the inhibition rate of drugs to tumor cells was calculated as percentage of dead cells, as described elsewhere [39 (link)]. The IC₅₀ values were obtained by fitting the data to a sigmoidal dose-response curve (variable slope) using Prism 4 software (GraphPad) [48 (link)]. Each experimental condition was repeated for at least three times.

ELISA was performed to determine the concentration of HGF in conditioned medium of hASCs and angiotensin II in conditioned medium of cardiac fibroblasts by using a human HGF ELISA kit (Neobioscience, Beijing, China) and angiotensin II ELISA kit (Phoenix Pharmaceuticals, Burlingame, CA, USA), respectively, following the manufacturer’s instruction. Absorbance of standards and samples at 450 nm were measured using a microplate reader (Multiskan G0, Thermo Scientific, Rockford, IL). Both standard curves with known concentration of HGF and angiotensin II versus absorbance at 450 nm were plotted. By referring to the standard curves, both the concentration of HGF in conditioned medium of hASCs and angiotensin II in conditioned medium of cardiac fibroblasts, which corresponds to its absorbance at 450 nm, was determined. Absorbance at 450 nm of medium without cell (free of angiotensin II and HGF) was used for normalization.

The DPPH assay was performed as described by Hidalgo et al. (2010) (link). The methanolic solution of DPPH is purple/violet colored, which fades to pale yellow in the presence of antioxidants, and the loss in absorbance is measured at 517 nm. A 100 μM DPPH solution was prepared in methanol, and 290 μl of this solution was mixed with 10 μl of individual compound/seed extract or their combinations. The reaction was carried out in a 96-well microplate, incubated in the dark at room temperature for 1hr, and absorbance was measured at 517 nm using a microplate reader (ThermoScientific Multiskan G0). The percentage DPPH radical scavenging activity was calculated by the following equation:
Where A_c is the absorbance of the control and A_s is the absorbance of the sample. Solution without the sample (seed extract or phytochemical) was taken as control. The results were expressed as EC₅₀ (μM) obtained by plotting a curve between concentration and inhibition percentage. EC₅₀ is the effective concentration necessary to get 50% inhibition. The lower the EC₅₀ value, higher will be the antioxidant activity.