Accuri 6 plus software

To evaluate the apoptosis rate of cells in the tumor microenvironment, SW480 cells were seeded at a density of 3.5 × 10⁵/well in a 6-well culture plate. The next day, cells were treated with MTX or Glu-MTX at a dose of 10 µM for 48 h. After treatment, cells were washed with PBS solution, detached by using 0.25% trypsin in EDTA, centrifuged, and prepared according to the manufacturer’s instructions from Annexin V-FITC PI Apoptosis Detection Kit (Abcam, Cambridge, UK). First, cells were suspended in 500 µL of 1× Binding Buffer. Afterward, 5 µL of Annexin V (Annexin V-FITC) and 1 µL of propidium iodide (PI, 50 µg/mL) were added to each sample. The samples were incubated for 20 min in darkness at room temperature. For the evaluation of apoptosis, a BD Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) was used. Control cells, MTX, and Glu-MTX-treated samples were measured in triplicate. The obtained results were analyzed using the BD Accuri 6 Plus Software (Becton, Dickinson, NJ, USA).

The ratio of live, early apoptotic, late apoptotic and dead cells was measured using Annexin V Apoptosis Detection Kit APC (Thermo Fisher Scientific Inc.). Cells were seeded at a density of 1 × 10⁶/well in 6-well culture plates and treated as described in experimental conditions with 6-shogaol with/without FU for 48 h. The cells were washed with PBS, detached from each well of 6-well plate by Gibco™ Trypsin-EDTA reagent and again washed with PBS. After centrifugation cell pellets from each tube were resuspended in 1 ml of 1XBinding Buffer. Then the amount of 100 μL of each cell sample was added to a new 1,5 ml tube and mixed with 5 μL of APC fluorochrome-conjugated Annexin V and 5 μL of Propidium Iodide Staining Solution. After 20 min of incubation at room temperature in the dark, samples were gently mixed and then loaded onto BD Accuri 6 Analyzer (Becton, Dickinson and Company, NJ, USA). Each sample was tested in triplicates. The results were analyzed using BD Accuri 6 Plus Software (Becton, Dickinson and Company).