Acridine orange propidium iodide

DAPI staining is used to assess apoptosis-related morphological changes such as nuclear fragmentation or condensation. The acridine orange/propidium iodide stain used in dual staining shows the apoptotic induction caused by the AgNPs treatment. AO stains both live and dead cells, whereas PI stains only the dead cells. In brief, 3 × 10⁵ of both A549 and BEAS-2B cells were seeded separately in the 6-well plates and treated with appropriate IC₅₀ values of AgNPs-GA, Cisplatin, and AgNPs-GA + Cisplatin for 24 hours at 37°C with 5% CO₂. Following incubation, the cells were washed with PBS and then fixed with 1 mL of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, Missouri, USA) and cold 70% ethanol for 10 minutes at room temperature. Then, the cells were stained with a 1 : 1 ratio of 10 µg/mL of DAPI (Nacalai, Tesque chemical, Kyoto, Japan) and 10 μg/mL acridine orange/propidium iodide (Sigma-Aldrich, St. Louis, Missouri, USA) and incubated for 30 minutes in the dark. An Olympus IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan) was used to observe and record the morphology of the cells.

Sodium dodecyl sulfate (SDS), 3-(4,5-dimethyltiazole-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 4′,6-diamidino-2-phenylindole (DAPI), Acridine Orange/Propidium Iodide and DMEM/Ham’s F12 medium were obtained from Sigma–Aldrich (Burlington, VT, United States). Fetal bovine serum (FBS) and Pen-Strep solution were purchased from Gibco (New York, NY, United States). Phosphate-buffered saline (PBS) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Prilocaine (3%), a local anesthetic drug, was obtained from Exir Pharmaceutical Company, Iran. Alkaline phosphatase kit was bought from Pars Azmoon (Tehran, Iran). DENAzist Column RNA Isolation Kit was obtained from DENAzist Asia Co., (Mashhad, Iran). cDNA synthesis kit was purchased from Parstous Co. (Tehran, Iran).