2.9. MTT cytotoxicity assay
The cytotoxicity of beetroot juice, alone or combined with fruit
juices (pH 3.5 MIX-es), towards HT29 cells was determined by MTT
assay. The exponentially growing cells were seeded in 96-well
tissue culture plates (about 25,000 cells/well in 0.15 mL). The
cells were allowed to settle for 24 h at 37 C, then they were treated
for 24 or 72 h with 0.05 mL of different concentrations ranging from
10 to 250 mL/L of tested plant samples diluted with PBS. In the case
of the shorter exposure, the medium was aspirated after 24 h from
the wells, replaced with fresh medium and the cells were incubated
at 37 C for further 48 h. After 72 h of the total incubation time,
0.05 mL of MTT solution (4 g/L) was added and cells were left for
another 4 h at 37 C. Finally, medium was carefully removed from
wells, and formazan crystals formed dissolved in 0.05 mL of DMSO.
The absorption of the resultant solutions was determined at
540 nm with TECAN Infinite M200 plate reader (Tecan Group Ltd.,
Switzerland). The cytotoxicity was expressed as growth inhibition
of cells exposed on tested plant samples compared to control nontreated
cells whose growth was regarded as 100%.