Ez read 2000

The non-cytotoxic concentration of the samples on DU-145 cells was determined by sulforhodamine B (SRB) assay, as previously described [50 (link)]. Human prostate cancer cells (1 × 10⁵ cells/mL) were cultured in 96-well plates for 24 h at 37 °C and 5% CO₂. The cells were tested with the samples in the range of 0.0001–10 mg/mL for another 24 h. After incubation, the adherent cells were fixed in situ by 50% trichloroacetic acid and dyed with 0.04% SRB solution, which was prepared in 1% acetic acid. The bound dye was solubilized, and the absorbance was measured at 515 nm using the microplate reader (EZ Read 2000, Biochrom, Holliston, MA, USA). The percentages of cell viability were calculated by Equation (4), where Abs_Control is the absorbance of cells cultured with the medium without supplementation, Abs_Blank is the absorbance of cells treated with the solvent, and Abs_Sample is the absorbance of cells treated with the sample: $$\mathrm{Cell}\mathrm{viability}\left(\%\right)=\frac{{\mathrm{Abs}}_{\mathrm{Sample}}-{\mathrm{Abs}}_{\mathrm{Blank}}}{{\mathrm{Abs}}_{\mathrm{Control}}-{\mathrm{Abs}}_{\mathrm{Blank}}} \times 100$$

The ABTS assay was evaluated according to the previous method with some modifications [50 (link)]. The assay was based on ABTS radical scavenging ability in comparison to that of Trolox and L-ascorbic acid in the range of 0.01–10 mg/mL. The ABTS stock solutions were prepared by placing 7 mM of ABTS radical solution in distilled water reacted with 2.45 mM of potassium persulfate solution, and they were stored at room temperature in the dark for 12–16 h. Then, the ABTS working solution was diluted to obtain an absorbance of 0.7–0.9 at 734 nm with distilled water using the microplate reader (EZ Read 2000, Biochrom, Holliston, MA, USA). The sample (25 µL) was reacted with 200 µL of ABTS solution for 10 min. The percentages of the ABTS radical scavenging activity were calculated by Equation (2), where Abs_Control is the absorbance of the ABTS solution, and Abs_Sample is the absorbance of the ABTS radicals that reacted with the sample: $$\mathrm{ABTS}\mathrm{radical}\mathrm{scavenging}\mathrm{activity}\left(\%\right)=\frac{{\mathrm{Abs}}_{\mathrm{Control}}-{\mathrm{Abs}}_{\mathrm{Sample}}}{{\mathrm{Abs}}_{\mathrm{Control}}}\times 100.$$
The concentration providing 50% scavenging activity (MC₅₀) (mg/mL) was obtained from the linear relationship between the different concentrations of the samples and the percentages of the ABTS radical scavenging activity.

The cytotoxicities of kombucha tea were tested using MTT assay. NIH/3T3 cells were used as normal cell control. Human colorectal carcinoma (Caco-2) and NIH/3T3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) that had been supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HyClone^TM, Pittsburgh, PA, USA), 100 Units/mL penicillin and 100 μg/mL streptomycin (CAISSON, Smithfield, UT, USA). After incubation at 37 °C in a 5% CO₂ incubator (SHEL LAB, USA), the cells were washed twice with phosphate buffer saline (PBS, pH 7.4) and trypsinized with 0.05% (v/v) trypsin-EDTA solution (CAISSON, USA). The Caco-2 and NIH/3T3 cells were plated in 96-well plates and incubated at 37 °C in a 5% CO₂ incubator for 24 h. After incubation, each concentration of kombucha tea was then added. The plates were incubated at 37 °C in a 5% CO₂ incubator for 48 h. The MTT solution (Bio Basic Inc., Amherst, NY, USA) was then added and the solution was incubated for 4 h. Finally, blue formazan crystals were dissolved with dimethyl sulfoxide and the absorbance was measured at 540 and 630 nm by micro plate reader (EZ Read 2000, Biochrom, Cambridge, UK). The percentage of cell viability was calculated by comparing the relevant values to the cell control [19 (link)].