Annexin 5 propidium iodide detection kit

The Annexin V/propidium iodide (PI) detection kit (BD Biosciences, PA, USA) was employed to quantify apoptosis using flow cytometry. H1299 and H1975 cells were seeded in each well of a 12-well plate at 2.5 × 10⁵ cells/well. After incubation for 24 h, the cells were treated with compound 15 at 0.5, 1 and 2 μM or PPT (1 μM) for 24 h. Then, the cells were collected and binding buffer (100 μL), FITC annexinV (5 μL), and propidium iodide (PI, 10 μL) (eBioscience, San Diego, CA, USA) were added to the cell suspension. The cells were gently vortexed and incubated at room temperature in the dark for 15 min before measurement by flow cytometry (BD FACSCalibur^TM) within 1 h.

At various time points, control and treated cells were collected following treatment and subjected to apoptosis measurement using the annexin V/propidium iodide (PI) detection kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer's protocol. A total of 10,000 cells (within whole-cell gates) per replica (3 independent experiments) were subjected to a flow cytometric analysis to evaluate the green fluorescence of annexin V and the red fluorescence of DNA-bound PI. All the data was analyzed by FlowJo software (BD Bioscience, San Jose, CA, USA).

RPMI8226 and NCI-H929 underwent seeding in a 6-well plate at 2.0 × 10⁵/well. Cells were treated for 48 h with DMSO, 30 μM TMZ (RPMI8226), 20 μM TMZ (NCI-H929), and 3 μM Nira, respectively, for both cell lines, or combined 3 μM Nira and 30 μM (RPMI8226) or 20 μM (NCI-H929) TMZ for 48 h. The Annexin V/propidium iodide (PI) detection kit (BD Pharmingen™) was utilized for apoptosis quantitation. In brief, after treatment with specific drugs for 48 h, the cells underwent incubation, shielded from light at ambient, with Annexin V/FITC and PI for 15 min. Analysis was performed flow-cytometrically with an Epics flow cytometer. After treatment for 48 h with the drugs, adding 5 μL Annexin V and 15 μL PI for each sample, and incubation in the dark for 15 min, apoptosis analysis was performed by flow cytometer. Cells that were Annexin V/FITC positive (with translocation of membrane phospholipid phosphatidylserine (ps) from the inner to the outer leaflet of the plasma membrane) and PI negative (with intact cellular membrane excluding PI) were regarded as early apoptotic cells, whereas positivity for both Annexin V/FITC and PI was considered as late apoptotic or necrotic cells.