Accuri c6 plus flow cytometer and software

MCF7, SKOV-3, and HeLa apoptotic cells were quantified using an Annexin V-FITC/PI apoptosis detection kit (Immunostep, Salamanca, Spain) according to the manufacturer’s guidelines. 1.5 mL/well of cells in the exponential growth phase at 2·10⁴ cells/mL were seeded onto 12-well plates and left to attach overnight. After 24 h incubation, cells were treated with selected compounds. After 72 h (MCF7, SKOV-3, and HeLa) and 6 days (MCF7) post-treatment, attached and floating cells of treated and untreated wells were collected, centrifuged, resuspended in Annexin V binding buffer, and stained with Annexin V-FITC/PI. Cells were then incubated in darkness at room temperature for 15 min and a total of 30,000 cells were acquired and analyzed using the BD Accuri™ C6 Plus Flow Cytometer and Software (version 1.0.264.21, BD Biosciences, San José, CA, USA), respectively. Compounds were dissolved in DMSO and the final solvent concentration never exceeded 0.5% (v/v). Control wells included cells with 0.5% (v/v) DMSO.

MCF7, HeLa, and HT-29 apoptotic cells were quantified using an Annexin V-FITC/PI apoptosis detection kit (Immunostep, Salamanca, Spain) according to the manufacturer’s guidelines. 1.5 ml/well of cells in the exponential growth phase at 2 × 10⁴ cells/mL were seeded onto 12-well plates and left to attach overnight. After 24 h incubation, cells were treated with compound 25 at 87.5 or 175 nM. 72 h post-treatment, attached and floating cells of treated and untreated wells were collected, centrifuged, resuspended in the Annexin V binding buffer, and stained with Annexin V-FITC/PI. Cells were then incubated in darkness at room temperature for 15 min and a total of 30,000 cells were acquired and analysed using the BD Accuri™ C6 Plus Flow Cytometer and Software (version 1.0.264.21, BD Biosciences, San José, CA, USA), respectively. Compounds were dissolved in DMSO and the final solvent concentrations never exceeded 0.5% (v/v). Control wells included cells with 0.5% (v/v) DMSO.