*2.11. Cytotoxicity Test of Kombucha Tea on Human Colorectal Carcinoma (Caco-2) and NIH*/*3T3 Cells*

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 (HyCloneTM, 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].
