Cycle phase determination kit

To analyze the underlying mechanism of the aspirin-mediated inhibition of tumor cell growth, flow cytometric analyses were performed using the Cycle Phase Determination kit (Cayman Chemical Co.). HCC Huh-7 cells (1.0×10⁶ cells/100-mm diameter dish) were treated with 2.5 mmol/l aspirin for 24 to 48 h. Cells were scraped and centrifuged to obtain the cell pellet, which was resuspended in phosphate-buffered saline (PBS) (10⁶ cells/ml). An equal volume of cell suspension was added to the cycle phase determination fixative and stored at −20°C until further analysis. For cell cycle analysis, the cells were suspended in 100 µl of PBS with 10 µl RNase A (250 µg/ml) and 10 µl propidium iodide (PI) stain (100 µg/ml), followed by incubation at room temperature in the dark for 30 min. Flow cytometry was performed to compare the proportion of aspirin-treated and control cells in each phase of the cell cycle. Flow cytometry was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter) with an argon laser (488 nm), and the percentages of cells were analyzed using Kaluza software version v2.1 (Beckman Coulter). The experiments were repeated thrice.

We conducted a flow cytometric analysis using the Cycle Phase Determination kit (Cayman Chemical Co.) to evaluate the mechanism of growth inhibition by Gal-9. CW-2 cells were digested with 0.25% trypsin and plated in 100-mm-diameter dishes at 1.0×10⁶ cells per dish. After incubation for 24 h without FBS, CW-2 cells were treated with 0.3 µM of Gal-9 or dimethyl sulfoxide (DMSO; control) for another 24 h, then harvested, washed with phosphate-buffered saline (PBS), suspended in 500 µl of PBS plus 10 µl of RNase A (250 µg/ml) and 10 µl of propidium iodide (PI) stain (100 µg/ml), and incubated for 30 min.
To determine the apoptosis rate of CW-2, CACO-2, and WiDr cells, we used flow cytometry and the Annexin V-FITC Early Apoptosis Detection Kit (Cell Signaling Technology, Inc.). CW-2, CACO-2 and WiDr cells were plated in 100-mm-diameter dishes at 1.0×10⁶ cells per dish and treated with 0.3 µM Gal-9 or DMSO control for 24 h. After incubation for 24 h, CW-2, CACO-2 and WiDr cells were harvested, and washed with PBS. Staining was performed according to the manufacturer's protocol. After adding Annexin V-FITC and PI, we analyzed apoptosis and necrotic cell death. Flow cytometry was conducted with a Cytomics FC 500 flow cytometer (Beckman Coulter) equipped with a 480-nm argon laser. Cell percentages were analyzed with Kaluza software version 2.1 (Beckmann Coulter).

Flow cytometric analyses were performed using the Cycle Phase Determination kit (Cayman Chemical Company). HuCCT-1 and TKKK cells (1.0×10⁶ cells/100-mm dish) were treated with 2.5 mmol/l aspirin or without for 24 to 48 h. Cells were trypsinized and resuspended in phosphate-buffered saline (PBS) at a density of 1×10⁶ cells/ml. Approximately 1×10⁶ cells were stained in 100 µl of PBS with 10 µl RNase A (250 µg/ml) and 10 µl propidium iodide (PI) stain (100 µg/ml) and incubated at room temperature in the dark for 30 min. Flow cytometry (FCM) was performed to compare the proportion of aspirin-treated and control cells in each phase of the cell cycle. FCM was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter, Inc.) with an argon laser (488 nm), and the percentages of cells were analyzed using the Kaluza software version v2.1 (Beckman Coulter, Inc.). The experiments were repeated thrice.