Apoptosis detection kit 2

Cells were resuspended in 400 μL of binding buffer containing 5 μL of annexin V FITC and 5 μL propidium iodide (Apoptosis Detection Kit II, BDBiosciences) for 15 min at room temperature. Annexin V binding was evaluated by flow cytometry (FACScalibur, BD Biosciences), and after acquisition of 30,000 events, the data were analyzed in CellQuest and FlowJo software.

To evaluate the mode of cell death induced by afzelin, a flow cytometry measurement was performed using Apoptosis Detection Kit II (BD Pharmingen, San Diego, CA, USA), according to the manufacturer’s instructions. Cells were trypsinised, resuspended in the medium, and then in the binding buffer. In the next step, they were stained with Annexin V and propidium iodide (PI) 15 min at room temperature (RT) in the dark place. As a negative control, the cells cultured in a drug-free medium were used. Optimal parameter settings were found using a positive control—cells incubated with 3% formaldehyde in buffer, for 30 min on ice. To analyse the results, FACSCanto II cytometer (BD, San Jose, CA, USA) with FACSDiva software (BD Biosciences systems, San Jose, CA, USA) was applied.

Sperm apoptosis was evaluated by Annexin V/PI staining using a commercially available kit (Apoptosis Detection Kit II; BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions, and as previously described^{15 (link),46 (link)}. Briefly, an aliquot of cells containing 1 × 10⁶ sperms/ml was resuspended in 100 µl binding buffer, labeled with 10 µl Annexin-FITC plus 10 µl PI, incubated for 15 min in the dark, followed by the addition of 400 µl binding buffer and immediately transferred to a flow cytometer and analyzed. The cell population of interest was gated on the basis of its forward and side-scatter properties. The different labeling patterns in the bivariate PI-Annexin V analysis that identified the different cell populations were FITC negative and PI negative and were designated as viable cells; FITC positive and PI negative as early apoptotic cells; FITC positive and PI positive as late apoptotic cells; and FITC negative and PI positive as necrotic cells. A total of 100,000 events were acquired for each sample in a flow cytometer and analyzed. Results are expressed as percentage of cells.

Cells were treated with different concentrations of ARV 825 for 48 h. Staining was performed using Apoptosis Detection Kit II (BD Biosciences, USA). Cells were harvested and washed twice with phosphate-buffered saline (PBS, Life technologies, USA), suspended in 1X binding buffer with 5 μl of FITC conjugated Annexin V and 5 μl of PI for 15 min in the dark at room temperature. Samples were analyzed using flow cytometric analysis (Sony SA3800). Cells positive for Annexin and PI were defined as apoptotic cells.

Annexin V-PI staining was performed using flow cytometric analysis as previously described^{50 (link)}. Briefly, 1 × 10⁶ cells were cultured with increasing concentrations of selinexor (0–1000) for 24 h. Staining was performed using Apoptosis Detection Kit II (BD Biosciences, USA). Cells were harvested and washed twice with phosphate-buffered saline (PBS; Life technologies, USA). Cells were suspended in 1X binding buffer containing 5 ul of FITCI conjugated Annexin V and 5 ul of PI for 30 min in dark. The samples were analyzed using LSR-II flow cytometer (BD, San Jose, CA, USA).

Irradiated and nonirradiated PC9 and A549 cells were cultured with different concentrations of cisplatin and bortezomib for 72 h for cell cycle analysis or 48 h for DNA damage analysis. Apoptotic cell death was assessed by double staining with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI; Apoptosis Detection Kit II, BD Pharmingen) as previously described^{22 (link)}. DNA damage was evaluated by staining cells with an anti-H2AX (pS139) antibody (BD Pharmingen). Fluorescence-activated cell sorting (FACS) data were acquired using a BD FACS Canto II (BD Biosciences, San Jose, CA). The mean fluorescence index was calculated using FlowJo software (TreeStar, Ashland, USA). Gating was implemented based on negative control staining profiles.

Cells were seeded in 6 well plates either in the presence or absence of different concentrations of inhibitors. A total of 48 h after seeding, staining was performed using Apoptosis Detection Kit II (BD Biosciences, New Jersey, USA). Cells were harvested and washed twice with phosphate-buffered saline (Thermo Fisher Scientific, Waltham, MA, USA), suspended in 1X binding buffer with 5 μL of Alexa Fluor 488 conjugated Annexin V and 5 μL of PI for 15 min in the dark at room temperature. A total of 10,000 events were captured per sample. Flow cytometric analysis was performed on a FACS LSR II flow cytometer. Cells positive for Annexin and PI were defined as apoptotic cells. Data were analyzed using FACSDiva software (BD Biosciences, NJ, USA).