Acridine orange 10 nonyl bromide nao

Cells were stained with 200 nM Mitotracker Green (Thermo Fisher Scientific Inc.) or Acridine Orange 10-nonyl bromide (NAO) (Sigma-Aldrich) harvested and analyzed by flow cytometry.

Calcium ionophore (A23187), benzamidine hydrochloride, N-acetyl-Leu-Glu-His-Asp trifluoro methylcoumarin (AC-LEHD-FMC), acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (AC-DEVD-AMC), sodium orthovanadate, dimethyl sulfoxide (DMSO), fluorescein isothiocyanate (FITC)-labeled annexin V, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester (CM-H2DCFDA), CHAPS, rhodamine 123, leupeptin hydrochoride, N-(2-Hydroxyethyl) piperazine-N′-ethanesulfonic acid (HEPES), fura-2/AM, monoclonal anti-phosphotyrosine antibody, acridine orange 10-nonyl bromide (NAO) and dithiothreitol (DTT) were from Sigma Chemicals, St. Louis (USA). Monoclonal anti-cytochrome c antibody and anti-β-actin were from Epitomics Burlingame, CA (USA). Anti-Caspase-3 antibody was from Santa Cruz Biotechnology, Inc. Texas (USA). Collagen type-I was from Chrono-log Corporation, Pennsylvania (USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1, 1-diphenyl-2-picrylhdrazyl (DPPH) were from HiMedia Laboratories, Mumbai (India). Lactate dehydrogenase (LDH) kit was from AGAPPE diagnostics Ltd., Kerala (India). γ-glutamyl p-nitroanilide and glycylglycine were from Sisco Research laboratories Pvt Ltd., Mumbai (India). All other reagents were of analytical grade.

WT HeLa cells were detached, centrifuged (200 g, 10 minutes), resuspended in DMEM and stained with 2 μM Acridine Orange 10-nonyl bromide (NAO, Sigma-Aldrich) at 37°C for 30 minutes in darkness (1,5 × 10⁶ cells/mL). After loading, cells were resuspended in HBSS (plus 1 μM CsA, 3 × 10⁵ cells/mL) and divided into identical aliquots. A Beckton Dickinson II flow cytometer was used, and 10,000 events were counted for each measurement. Excitation was at 488 nm and fluorescence was collected in the 542–585 nm interval. Data were analyzed using the BD VISTA software. Averages ± s.d. of the medians of fluorescence distribution histograms were plotted, normalized to the value measured for the control.