Trypan blue stain 0.4

HT29 cells, kindly obtained from the Northern Ireland Center for Food and Health, were seeded in six well plates (2 mL/well) at the density of 2 × 10⁵ cell/well. Cells were treated with 10%, 25%, and 50% of spinach juice for 24 h and then counted. Each experiment was performed in triplicate. For cell counting, after 24 h of treatment, the cells were trypsinized and re-suspended in DMEM supplemented with 1% glutamine, 1% penicillin/streptomycin, and 10% fetal bovine serum (all purchased from Lonza, Vievers, Belgium) with a sterile 1 mL pipette. In addition, 30 µL of cell suspension were mixed with the same amount of trypan blue (Trypan Blue Stain 0.4%—Lonza, Walkersville, MD 21793 USA). Cells were visually examined at 400× magnification to determine whether they took up or excluded dye. A viable cell presents a clear cytoplasm whereas a nonviable cell shows a blue cytoplasm. For each sample, 100 cells were scored.

Trypan blue exclusion assay was used to assess the viability and the count of cells at the beginning and at the end of each passage (P0–P2), and prior to transplantation. Trypan Blue stain 0.4% (Lonza, Basel, Switzerland) and hemocytometer chambers (MARIENFIELD, Thuringia, Germany) were used for this purpose. Cells of P2 were washed after trypsinization with phosphate-buffered saline (PBS), (Lonza, Basel, Switzerland), and suspended in DMEM, and adjusted to be 4 × 10⁶ cells/mL Each rat of group 6 received 1 mL of this suspension by IV injection in the tail vein.