Modfit lt software version 4

The HK1 cells were seeded into 100-mm culture plates at a density of 4.0×10⁵ cells/dish in 6 ml culture medium. The cells were treated with GnRH at a concentration of 10^-9 M for 48 h. Untreated cells were used as a control. The cells were prepared for cell-cycle analysis using a CycleTEST PLUS DNA Reagent kit (BD BioSciences, San Jose, CA, USA). A pellet containing 5×10⁵ cells was gently resuspended in 250 μl solution A, containing trypsin (BD Biosciences), followed by 200 μl solution B, containing trypsin inhibitor and RNase A (BD Biosciences), and incubated at room temperature for 10 min each. A total of 200 μl propidium iodide (PI) was added and the cell suspensions were incubated at 4°C in the dark for 10 min. Flow-cytometric analysis of the cellular DNA content was performed using Cell Quest Pro software version 6.0 (BD Biosciences) on a FACS Calibur flow cytometer (BD BioSciences) and the results were analysed using ModFit LT™ software version 4.0 (Verity Software House, Inc., Topsham, ME, USA).

The CFPAC-1 cells were placed in 6-well plates at 5×10⁴ cells/well and treated with NS or MET (10, 20 or 40 mmol/l). Separate cell cultures were collected and trypsinized 48 h later, then washed twice with cold phosphate-buffered saline (PBS). The cells were subsequently incubated with an annexin V/PI double staining solution (Sigma-Aldrich) at room temperature. Fifteen min later, the stained cells were analyzed by flow cytometry and the percentage of apoptotic cells (those in the lower right quadrant) was calculated with ModFit LT software version 4.0 (Verity Software House, Inc., Topsham, ME, USA). Cell cycle analysis was performed by flow cytometry using CellQuest Pro software version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA). In combination therapy experiments, cells were treated with MET (20 mmol/l) and GEM (5 µmol/l), alone or in combination for 48 h. The percentage of apoptotic cells was calculated as above.