Acridine orange

Unless specifically stated, the chemicals used in this study were purchased from Roth (Karlsruhe, Germany) and Merck (Darmstadt, Germany) and were of analytical grade. Acridine orange was obtained from Polysciences Europe GmbH (Eppelheim, Germany) and Merocyanin 540 (M540) and YoPro-1 from Molecular Probes (Leiden, the Netherlands). The fluorescent dyes fluorescein-isothiocyanate conjugated peanut agglutinin (FITC-PNA) and Pisum sativum agglutinin (FITC-PSA) were purchased from Axxora (Lörrach, Germany). Propidium iodide (PI) and Rhodamine 123 (R123) were obtained from Sigma-Aldrich (Steinheim, Germany).

Acidic intracellular compartments were evaluated using acidic vesicular organelle (AVO) staining. U937 cell staining was performed as previously described [11 (link),12 (link),13 (link)]. To examine the degree of autophagy in U937 cells, the cells were treated with hesperetin (0, 12.5, 25, 50, and 100 µM) for 24 h. The cell pellets were washed twice with cold PBS and centrifuged at 1200 rpm for 5 min at 4 °C. Acridine orange (Polysciences, Warrington, PA, USA) was added at a final concentration of 1 mg/mL for 15 min at 37 °C in the dark. The FACSCalibur system (Becton Dickinson) was used to analyze the autophagic cells. Cell viability assay was used to evaluate the inhibition of cell proliferation potentials of 3-Methyladenine (3-MA) and bafilomycin A1 (Baf-A1). Also, U937 cells were seeded in 96-well plates (4 × 10⁴ cells/well) and incubated at 37 °C for 24 h, pretreated with or without 3-MA (2 mmol/L) for 2 h, and then stimulated with various concentrations of hesperetin (0, 25, 50, and 100 µM). The wells without 3-MA or Baf-A1 and hesperetin addition were used as controls. Each concentration was replicated three times. Following incubation for 24 h, 10 µL of CCK-8 (Sigma-Aldrich) was added to each well, and the cells were reincubated at 37 °C for 2 h. Absorbance was measured using a multifunctional microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm.