Accuri c6 analysis software

To investigate the effects of Oxy on MCF-7aro cell cycle progression, the DNA content was assessed by flow cytometry. MCF-7aro cells (7 × 10⁵ cells/mL) stimulated with T (1 nM) were incubated with Oxy (1 and 2.5 µM) for 3 days. Cells only treated with T were considered as control. After the incubation period, cells were fixed with 70% cold ethanol and stained with a DNA staining solution (5 µg/mL Propidium Iodide (PI), 0.1% Triton X-100 and 200 µg/mL DNase-free RNase A (Sigma-Aldrich Co., Saint Louis, MI, USA)), as previously described [19 (link)]. DNA content was analyzed by flow cytometry based on the acquisition of 40 000 events in a BD Accuri™ C6 cytometer (San Jose, CA, USA), equipped with a BD Accuri™ C6 analysis software. Detectors for the three fluorescence channels (FL-1, FL-2 and FL-3) and for forward (FSC) and side (SSC) light scatter were set on a linear scale. Debris, cell doublets and aggregates were gated out using a two-parameter plot of FL-2-Area to FL-2-Width of PI fluorescence. Data were analyzed using the BD Accuri™ C6 analysis software. The anti-proliferative effects were indicated by the percentage of cells in G₀/G₁, S and G₂/M phases of the cell cycle.

To study the effects of G on MCF-7aro cell cycle progression, the DNA content was assessed through labeling with propidium iodide (PI) and by performing flow cytometry. Cells (7 × 10⁵ cells/mL) stimulated with T (1 nM) were incubated with G (1 µM), Exe (10 µM), or with G plus Exe, for 3 days. Cells only treated with T were considered the control group. Following treatments, cells were fixed with cold 70% EtOH and stained with a DNA labeling solution (Propidium Iodide at 5 μg/mL, 0.1% Triton X-100 and 200 μg/mL DNase-free RNAse A) (Sigma–Aldrich Co., Saint Louis, MO, USA), as previously described [39 (link)]. DNA content was analyzed by flow cytometry based on the acquisition of 40,000 events in a BD Accuri C6 cytometer (San Jose, CA, USA). Detectors for the three fluorescence channels (FL-1, FL-2, and FL-3) and for forward (FSC) and side (SSC) light scatter were set on a linear scale. Debris, cell doublets, and aggregates were gated out using a two-parameter plot of FL-2-Area to FL-2-Width of PI fluorescence. Data were analyzed using the BD Accuri™ C6 analysis software. The antiproliferative effects were indicated by the percentage of cells in the G₀/G₁, S, and G₂/M phases of the cell cycle.

NET cells were grown and treated with WNT974 (1–16 µM) for 72 h and then harvested through trypsinization, centrifugation and two washing steps in phosphate-buffered saline (PBS). After that, the cells were re-suspended in Nicoletti solution containing propidium iodide (Sigma-Aldrich, Taufkirchen, Germany) and then analyzed with the BD Accuri C6 flow cytometer and quantified using BD Accuri C6 Analysis software (BD Biosciences, Heidelberg, Germany) for cell cycle distribution. All experiments, consisting of technical triplicates, were repeated at least three times.