Annexin 5 binding buffer 1

Tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Alexa Fluor 488 conjugated goat anti-mouse IgG cross-adsorbed secondary antibody (2 mg/mL, Cat #A32723) and paraformaldehyde were obtained from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Phosphorylated EGFR monoclonal IgG1 mouse antibody (15A2:sc-81488) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Annexin V binding buffer 1×, APC Annexin V, 7-Amino-Actinomycin D (7-AAD) and PI/RNase staining buffer were obtained from BD Biosciences (San Jose, CA, USA). The secondary goat anti-mouse IRDye secondary antibody was purchased from LI-COR Biotechnologies (Lincoln, NE, USA), and the Vectashield Antifade Mounting medium with DAPI (H-1200-10) was purchased from Vector Laboratories (Burlingame, CA, USA). CIEAs were provided by Dr. Halaweish (South Dakota Ste University, Brookings, SD, USA). All other chemicals used were of analytical grade.

Apoptosis was studied by labeling apoptotic cells with Annexin V fluorescein isothiocyanate (BD Biosciences Pharmigen, CA, USA) and necrotic cells with propidium iodide (PI). After ATO treatment for 48 hours (2, 4 or 8 μM), the cells were trypsinized, washed with ice-cold PBS 1X and resuspended in 200 μL of Annexin V binding buffer 1×(BD Biosciences Pharmigen, CA, USA). Cells were labeled with 5 μL of Annexin V and 50 μL of PI solution (50 μM). 10.000 events per treatment were analyzed with a BD FACS Calibur ™ flow cytometer (BD Biosciences Pharmigen, CA, USA). Three independent experiments were performed in triplicate.

The apoptosis-inducing effects of the CIEAs were evaluated using flow cytometry as previously described [55 (link)]. Briefly, cells were plated at a density of 1 × 10⁵ cells/well in a 6-well plate (Corning, NY, USA) and challenged with IC₅₀ concentrations of the individual CIEAs for 48 h at 37 °C. The cells were then harvested, washed three times with 1× PBS (Corning, NY, USA) and resuspended in Annexin V binding buffer 1× (BD Biosciences, San Jose, CA, USA). The cells were then stained with APC Annexin V (BD, USA) and 7-AAD (BD Biosciences, San Jose, CA, USA), incubated in the dark for 25 min and analyzed with an Accuri C6 Pus flow cytometer (BD, Biosciences, San Jose, CA, USA).