Annexin 5 fluorescein isothiocyanate and propidium iodide

MCF-7 cells were seeded in 6-well plates at 1.2 × 10⁵ cells/well, whereas CCRF-CEM and K-562 cells were seeded in 24-well plates at a density of 2 × 10⁵/mL. Cells were treated with the tested compounds used in 5 µM and 10 µM concentrations. Then, the plates were incubated for 48 h. After exposure to the examined compounds, the cells were collected and centrifugated at 200× g at 4 °C for 5 min, washed twice in cold phosphate-buffered saline (PBS), and subsequently suspended in binding buffer at 1 × 10⁶ cells/mL. Subsequently, 100-μL aliquots of the cell suspension were labeled according to the instructions of the respective manufacturer’s kit. Briefly, annexin V-fluorescein isothiocyanate and propidium iodide (BD Biosciences, Pharmingen, San Diego, CA, USA) were added to the cell suspension, and the mixture was vortexed, then incubated for 15 min at RT in the dark. A cold binding buffer (400 μL) was then added, and the cells were vortexed again and kept on ice. Flow cytometric measurements were performed within 1 h after labeling. Viable, necrotic, early, and late apoptotic cells were detected by flow cytometry using a BD FACSCanto II flow cytometer and analyzed using BD FACSDiva operating software (BD Biosciences, San Jose, CA, USA).

Analysis of apoptosis by flow cytometry was measured after cells were treated for 72 hours. Cells were washed and double stained with annexin V‐fluorescein isothiocyanate and propidium iodide (BD Biosciences) followed by flow cytometry analysis. The mitochondrial transmembrane potential (ΔΨ_m) was determined using JC‐1 Mitoscreen (BD Biosciences). Cells were harvested after the indicated treatment and stained with JC‐1, washed, and subjected to detection by flow cytometry. Detection of JC‐1 aggregates was dependent on JC‐1 concentration, where higher concentrations indicated accumulation of JC‐1 aggregates within polarized (intact) mitochondria membrane. All flow cytometric analysis were performed using a BD LSR II flow cytometer and data were analyzed using FlowJo software (Becton, Dickinson and Company).

The single-cell suspension of NSCLC cells was prepared and fixed by 75% ethanol at 4°C for 12 h. The binding buffer containing Annexin V-fluorescein isothiocyanate and propidium iodide (BD Biosciences, San Diego, CA, USA) was used to stain cells in the dark at room temperature for 15 min, following by apoptosis assay under the flow cytometry (Becton Dickinson, San Jose, CA, USA). For cell cycle measurement, transfected NSCLC cells or control was collected and then fixed by 75% ethanol at 4°C for 12 h. After that, cells were incubated with 1 mL of PI/TritonX-100 staining solution containing RNase A for 30 min. flow cytometry (Becton Dickinson) was used examine cell cycle, and ModFit software (Becton Dickinson) was used for data analysis.

MCF-7 cells were seeded in six-well plates at 1.5 × 10⁵ cells/well. After 24 h of incubation, cells were treated with the tested compounds used in concentrations corresponding to their EC₅₀ values. Then, the plates were incubated for 24 h or 72 h. After exposure to the examined compounds, the floating cells were collected, and monolayer cell culture was trypsinized. The cells were centrifugated at 200× g at 4 °C for 5 min, washed twice in cold phosphate-buffered saline (PBS), and subsequently suspended in binding buffer at 1 × 10⁶ cells/mL. Subsequently, 100-μL aliquots of the cell suspension were labeled according to the kit manufacturer’s instructions. Briefly, annexin V-fluorescein isothiocyanate and propidium iodide (BD Biosciences, Pharmingen, San Diego, CA, USA) were added to the cell suspension, and the mixture was vortexed and then incubated for 15 min at RT in the dark. A cold binding buffer (900 μL) was then added and the cells were vortexed again and kept on ice. Flow cytometric measurements were performed within 1 h after labeling. Viable, necrotic, early, and late apoptotic cells were detected by flow cytometry using CyFlow Cube 8 (Sysmex, Norderstedt, Germany) flow cytometer.

A density of 1 × 10⁶ target cells was plated into six-well plates and treated with CDDP (3 μg/ml), NU7026 (2 μM) or 0.1% DMSO as a control for 24 h. They were then labeled with annexin-V-fluorescein isothiocyanate and propidium iodide (BD Biosciences, Franklin Lakes, NJ, USA) as previously described [54 (link)].