Annexin 5 fitc pi apoptosis kit

The cells undergoing apoptosis process were determined using Annexin V-FITC/PI apoptosis kit (ImmunoTools, Gladiolenweg 2, Friesoythe, Germany). NSCLC cells were seeded in 24-well plate with a density of 5 × 10⁴ cells/well for overnight and then treated with several concentrations of NB and NB-acid (0, 1, 5, 10, and 25 µM) for 24 h. After that, the cells were collected and suspended in 100 µL of annexin V-FITC binding buffer. Then, annexin V-FITC and PI were added and incubated for 15 min at room temperature as recommended in the manufacturer’s protocol (ImmunoTools, Gladiolenweg 2, Friesoythe, Germany). Guava easyCyte flow cytometer (EMD Millipore, Hayward, CA, USA) was used to analyze the cells.

Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), l-glutamine, antibiotic-antimycotic, trypsin-EDTA, and phosphate-bueredsaline (PBS) for cell culture were obtained from Gibco (Grand Island, NY, USA). Hoechst 33342, propidium iodide (PI), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), crystal violet, LY294002, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich, Co. (St. Louis, MO, USA). RIPA lysis buffer, primary antibodies (Mcl-1 (#94296), PARP (#9532), Bax (#5023), Bcl-2 (#4223), Akt (#9272), p-Akt (Ser473, #4060), c-Myc (#18583), Nanog (#4903), and β-actin (#4970), and secondary antibodies (anti-rabbit IgG (#7074) were obtained from Cell Signaling Technology (Danvers, MA, USA). CD133 primary antibody (ab19898) was acquired from Abcam (Waltham, MA, USA). Annexin V-FITC/PI apoptosis kit was purchased from ImmunoTools (Gladiolenweg 2, Friesoythe, Germany).

Annexin V-FITC/PI apoptosis kit (ImmunoTools, Gladiolenweg 2, Friesoythe, Germany) was introduced to investigate apoptotic cell death by using flow cytometry with annexin V-FITC/PI staining. NSCLC cells (H460 and H23) were seeded into 24-well plates at a density of 5 × 10⁴ cells/well and incubated overnight. The cells were treated with several concentrations of DH_25 (0–50 µM) and incubated for 24 h at 37 °C. After that, the cells were detached from the surface by trypsin-EDTA (0.25%). The cells were collected and incubated with 5 µL of annexin V-FITC and 1 µL of PI for 15 min at room temperature in the dark. Then, the cells were analyzed using GuavaCyte flow cytometry systems (GuavaSoft Software version 3.3, Merck, Burlington, MA, USA).