Fcs4 express software

The ECC-1 and KLE cells were treated with ONC201 for 24 to 36 h in six well plates. For apoptosis analysis, the cells were collected and resuspended in 100 μl binding buffer containing Annexin V-FITC and 0.5 μl of propidium iodide for 15 min. For cycle analysis, the cells were harvested and fixed in a 90% methanol solution for 1 h. The cells were resuspended in RNase A solution for 30 min, followed by incubation with propidium iodide (PI) staining solution for 10 min. All samples were immediately measured by Cellometer (Nexcelom, Lawrence, MA) to identify apoptotic cells and assess cell cycle progression [22 (link), 28 (link)]. The results were analyzed by FCS4 express software (Molecular Devices, Sunnyvale, CA).

The cells were grown in in 6-well plates overnight and then incubated with varying concentrations of ONC201 or vehicle control for 36 hours. Cells were harvested, washed with PBS, fixed in 2 ml of ice-cold 90% ethanol and stored overnight at -20°C until cell cycle analysis was performed. On the day of analysis, the cells were resuspended in 100 ul RNase A solution containing propidium iodide (2 mg/ml) for 30 min at room temperature in the dark. DNA content was determined by Cellometer (Nexcelom, Lawrence, MA). Cell cycle was analyzed using FCS4 express software (Molecular Devices, Sunnyvale, CA).

Simvastatin induced apoptosis was detected with the Annexin V FITC kit (Biolegend, San Diego, CA) on the Cellometer (Nexelom, Lawrence, MA). Briefly, 2 × 10⁵ cells/well were seeded into 6-well plates, incubated overnight and then treated with simvastatin at different doses for 24 h. The cells were then collected, washed with PBS and resuspended in 100 ul binding buffer. Subsequently, 1 ul of annexin V-FITC (100 ug/ml) and 0.5 ul of propidium iodide (2 mg/ml) were added in the binding buffer and placed in the dark for 15 minutes. The samples were immediately measured by Cellometer. The results were analyzed by FCS4 express software (Molecular Devices, Sunnyvale, CA). All experiments were performed in triplicate to assess for consistency of response.

The effect of glutamine on cell cycle progression was assessed by using Cellometer (Wang et al. 2014 (link); Nexcelom, Lawrence, MA, USA). Cells were plated at a density of 1.5×10⁵ cells/well in six-well plates overnight, and then treated with various concentrations of glutamine for 48 h. Cells were collected by 0.05% Trypsin (Gibco), washed with PBS solution, fixed in a 90% methanol solution and then stored at −20 °C until cell cycle analysis was performed. On the day of analysis, the cells were washed with PBS and centrifuged, resuspended in 50 μl RNase A solution (250 μg/ml) with 10 mM EDTA, followed by incubation for 30 min at 37 °C. After incubation, 50 μl of propidium iodide (PI) staining solution (2 mg/ml PI, 0.1 mg/ml azide, and 0.05% Triton X-100) was added to each tube and incubated for 10 min in the dark. The cell cycle was detected by Cellometer. The cell cycle progression was analyzed by the FCS4 Express Software (Molecular Devices, Sunnyvale, CA, USA).

The effect of NT1014 on cell cycle progression was measured using Cellometer (Nexcelom, Lawrence, MA). Briefly, the IGROV1 and SKOV3 cells were plated at 2.5 × 10⁵ cells/well in six-well plates and incubated overnight. Plates were then treated with NT1014 (from 0.1 to 1000 μM) for 24 h. The cells were harvested by trypsin digestion and washed with phosphate-buffered saline (PBS), before being re-suspended and fixed in 90 % pre-chilled methanol and stored at −20 °C overnight. The cells were treated with 50 μl RNase A solution (250 μg/ml, 10 mM EDTA) for 30 min at 37 °C and then stained with 50 μl of staining solution (containing 2 mg/ml propidium iodide (Hayward, MA), 0.1 mg/ml azide, and 0.05 % Triton X-100). The final mixture was incubated for 15 min in the dark before being analyzed by Cellometer. The results were analyzed using FCS4 express software (Molecular Devices, Sunnyvale, CA). The experiments were performed in triplicate and repeated three times for assessment of consistency.

The effect of phenformin on cell cycle progression was assessed using Cellometer (Nexcelom, Lawrence, MA). Cells were plated at a density of 2.5 x10⁵ cells/well in 6-well plates overnight and then treated with varying concentrations of phenformin for 24 hours. Cells were collected by 0.05% trypsin (Gibco, Grand Island, NY), washed with phosphate-buffered saline (PBS) solution, fixed in a 90% methanol solution and then stored at -20°C until cell cycle analysis was performed. On the day of analysis, the cells were washed with PBS and centrifuged, re-suspended in 50 ul RNase A solution (250 ug/ml) with 10 mM EDTA, followed by incubation for 30 min at 37°C. After incubation, 50 μl of propidium iodide (PI) staining solution (2 mg/ml PI, 0.1 mg/ml Azide and 0.05% Triton X-100) was added to each tube, and the cells were incubated for 10 min in the dark. The cells were then assessed by Cellometer. The results were analyzed using FCS4 express software (Molecular Devices, Sunnyvale, CA). Each experiment was performed in triplicate to assess for consistency of results.