Modfit lt v3

Apoptotic cells were detected using an Annexin V-FITC/PI kit (Dojindo Molecular Technologies, Inc.) and quantified using a BD LSRFortesa (BD Biosciences); data were analyzed using BD FACSDiva Software (version 6.2; BD Biosciences). hFOB 1.19 cells were seeded in 6-well plates at a density of 1×10⁵ cells/well, and then the four different groups of cells were transfected for 48 h and treated with 4 mM melatonin for 48 h at 37°C. The treated cells were digested with 0.25% trypsin (without EDTA), centrifuged at 300 × g for 3 min at 4°C and then washed with cold PBS twice. After the supernatant had been discarded, cells were resuspended in 500 µl binding buffer, and Annexin-V-FITC (5 µl) and PI (5 µl) were added and mixed. The suspensions were incubated in dark for 20 min at room temperature prior to flow cytometric analysis. Finally, cells were analyzed using a BD FACScan flow cytometer equipped with ModFit LT v3.0 (Becton, Dickinson and Company) within 1 h. Cells were classified as follows: Viable (Annexin V-FITC-/PI-), early apoptotic (Annexin V-FITC+/PI-), late apoptotic (Annexin V-FITC+/PI+) or necrotic (Annexin V-FITC-/PI+). The experiment was repeated three times.

SKOV3, HeyA8 and A2780 cells were transfected with miR-508-3p, miR-ctrl, siRNA-ctrl or siRNA-CCNA2. At 48 h post-transfection, the cells were harvested by trypsinization, washed with PBS and fixed in ice-cold 70% ethanol at 4°C overnight. The cells were then washed in PBS and incubated with 50 μl 100 g/ml ribo-nuclease (Thermo Fisher Scientific, Inc.) for 20 min at room temperature to ensure that only the DNA was stained. Then, the cells were incubated in 200 μl 50 g/ml propidium iodide for 30 min at 37°C in the dark. Cell cycle distribution was assayed using a FACSCalibur™ Flow Cytometer (BD Biosciences). The data were analyzed with the ModFit LT v3.3 software (BD Biosciences).

DNA index of ADSC and Jurkat cells was measured with CycleTEST^™ PLUS DNA Reagent Kit (BD) using BD FACSCanto^™ flow cytometer and ModFit LT v3.3 software (version 3.3) (BD). The procedure was carried out on freshly thawed cells according to manufacturer’s instructions.

Cells (2 × 10⁵/3 mL) were treated with drug at the indicated concentration or DMSO for 24 h. For cell cycle analysis, after being washed twice with ice-cold PBS, cells were fixed in 70% cold ethanol for 4 h at 4 °C. ROS production was detected using the fluorescence probe 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluoresceindiacetate (carboxy-DCFDA) [24 (link)]. For apoptosis evaluation [23 (link)], cells were stained with Annexin V and PI (1 μg/mL), counted on a BD FACSAria flow cytometer, and analyzed by ModFitLT V3.0 software program (Becton Dickinson, Germany).

Flow cytometry analysis was determined using the annexin V/PI method (annexin V-FITC/PI apoptosis kit; BestBio) in accordance with the manufacturer's protocols. The cells were seeded into 25-ml flasks and divided into groups according to treatment. After 48 h, the cells were washed, harvested, resuspended in 100 µl 1X binding buffer and stained with 5 µl FITC-annexin V and 5 µl PI for 15 min at room temperature in the dark. The samples were analyzed via flow cytometry (Excitation=488 nm; Emission=530 nm). The percentage distributions of normal, early apoptotic, late apoptotic and necrotic cells were calculated using ModFitLT v3.0 software (BD Biosciences) and the results were calculated from three independent experiments.