title: Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry.
abstract: BACKGROUND
  The aim of this study was to evaluate the ability of dual acridine orange/ethidium bromide (AO/EB) staining to detect tumor cell apoptosis. According to apoptosis-associated changes of cell membranes during the process of apoptosis, a clear distinction is made between normal cells, early and late apoptotic cells, and necrotic cells.
  
  
  METHODS
  We cultured human osteosarcoma cells with 30, 60, and 120 µg/ml kappa-selenocarrageenan. To assess the rates of cell proliferation and apoptosis, cells were fluorescently stained with acridine orange/ethidium bromide (AO/EB) or stained with propidium iodide (PI) and analyzed by flow cytometry. All experiments were repeated at least 3 times.
  
  
  RESULTS
  Normal tumor cells, early and late apoptotic cells, and necrotic cells were examined using fluorescent microscopy. Early-stage apoptotic cells were marked by crescent-shaped or granular yellow-green acridine orange nuclear staining. Late-stage apoptotic cells were marked with concentrated and asymmetrically localized orange nuclear ethidium bromide staining. Necrotic cells increased in volume and showed uneven orange-red fluorescence at their periphery. Cells appeared to be in the process of disintegrating. The percentage of apoptotic osteosarcoma cells detected by dual acridine orange/ethidium bromide (AO/EB) staining was not significantly different from that detected using flow cytometry (P>0.05).
  
  
  CONCLUSIONS
  Our results suggest that dual acridine orange/ethidium bromide staining is an economic and convenient method to detect apoptosis in tumor cells and to test tumor chemosensitivity compared with flow cytometry.
authors: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai, China (mainland).
authors: Kuan
authors: K
authors: Liu
authors: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai, China (mainland).
authors: Peng-cheng
authors: PC
authors: Liu
authors: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai, China (mainland).
authors: Run
authors: R
authors: Liu
authors: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai, China (mainland).
authors: Xing
authors: X
authors: Wu
mesh_terms: D000165:Acridine Orange; D017209:Apoptosis / Q000502:physiology*; D002351:Carrageenan; D049109:Cell Proliferation / Q000502:physiology*; D004996:Ethidium; D005434:Flow Cytometry; D006801:Humans; D016566:Organoselenium Compounds; D012516:Osteosarcoma / Q000503:physiopathology*; D011419:Propidium; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D016566:Organoselenium Compounds; C079969:kappa-selenocarrageenan; D011419:Propidium; D002351:Carrageenan; D004996:Ethidium; D000165:Acridine Orange