Cellquest pro software version 5

During the last 2 h of the final challenge, a blood sample was collected from all mice. The spleen was removed following sacrifice and 4 ml red blood cell lysis buffer was added prior to the tissues being ground. The mixtures were filtered and collected by a 75-µm sieve. Red blood cell lysis buffer was added, centrifuged (2,120 × g, 5 min, room temperature) and the supernatant was decanted. Subsequently, 10% RPMI-1640 (1 ml) (Thermo Fisher Scientific Inc.) was added and mixed with lymphocyte separation medium. The lymphocyte layer was aspirated, diluted to 10 ml by PBS and washed it to form a cell suspension. The cell concentration was adjusted with PBS to 1–5×10⁶ cells/ml. Samples were then stained with 0.5 µl antibodies: Anti-mouse ICOS-FITC (1:200), anti-mouse CXCR5-PE (1:200), anti-mouse CD4-PE Cy5 (1:200) and anti-mouse CD3 PE APC (1:100) (away from light at 4°C, 20 min). Cell pellets were resuspended in 500 µl FACS-PBS and maintained on ice prior to flow cytometry analysis. Flow cytometry was conducted on a BD FACSCalibur flow cytometer and analyzed using Cell Quest Pro software version 5.1 (both from BD Biosciences, Franklin Lakes, NJ, USA).

MGC-803 and SGC-7901 cells were harvested following caffeine treatment. Double staining with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI) was performed using an FITC-Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. GC cells were analysed using a FACScan flow cytometer equipped with CellQuest Pro software version 5.1 (both from BD Biosciences). The cells were assessed as viable, dead, early apoptotic and late apoptotic, and the relative amounts of early apoptotic cells were compared with those of the control groups. Cells used for cell cycle analysis were stained with PI using a Cycletest^® Plus DNA reagent kit (BD Biosciences), according to the manufacturer's protocol, and analysed using a FACScan flow cytometer. The percentages of cells in G0-G1, S and G2-M phase were counted and compared with those of the control groups.

HCT-116 and p53^−/− HCT-116 cells (1×10⁵ cells/ml) were cultured in 6-well plates and pre-treated with 20 nM bortezomib for 6 h and then with 20 µM celecoxib for an additional 18 h. For comparison, cells were treated either with 100 nM bortezomib or 80 µM celecoxib alone or co-treated (20 nM bortezomib + 20 µM celecoxib) for 24 h. Finally, cells were pre-exposed to 20 µM celecoxib for 6 h and then treated with 20 nM bortezomib for 18 h.
The percentages of cells undergoing apoptosis were determined by flow cytometry using fluorescein isothiocyanate (FITC)-labeled annexin-V and 7-aminoactinomycin (7-AAD) (both BD Biosciences, San Diego, CA, USA). Cells were harvested, rinsed with PBS, and resuspended in 100 µl of 1X annexin-V binding buffer (BD Biosciences). A total of 3 µl annexin-V-FITC and 3 µl 7-AAD were added, and cells were incubated at room temperature for 15 min in the dark, with gentle vortexing. The stained cells were analyzed using a FACSCalibur flow cytometer equipped with CellQuestpro software version 5.1 (BD Biosciences). Dot plot graphs were produced to quantify the percentage of viable cells (annexin-V⁻/7-AAD⁻), early-stage apoptotic cells (Annexin-V⁺/7-AAD⁻), late-stage apoptotic cells (annexin-V⁺/7-AAD⁺) and necrotic cells (Annexin-V⁻/7-AAD⁺).

Since Rho 123 is the substrate of ABCB1, its fluorescence intensity can be used as an indicator of the relative levels of ABCB1 (15 (link),21 (link)). A total of 2x10⁵ cells from the 4 cell lines per well at the logarithmic growth phase were seeded into a six-well plate. After 24 h incubation at 37˚C, the cells were exposed to different concentrations of OA (10, 20, 40, 80 and 100 µM) for 1 h at 37˚C. Rho 123 (5 µg/ml) was then added to the culture medium and the plate was incubated for 1.5 h at 37˚C. The total incubation time with OA was 2.5 h. The cells were then collected after centrifugation at 800 x g for 5 min at room temperature. The fluorescence intensity was detected using a BD FACSCalibur™ flow cytometer (BD Biosciences). The data were analyzed using CellQuest Pro software version 5.1 (BD Biosciences).