A Low-Cost Flow Cytometric Assay for the Detection and Quantification of Apoptosis Using an Anionic Halogenated Fluorescein Dye

Meyer, Mervin; Essack, Magbubah; Kanyanda, Stonard; Rees, D. J. G.

doi:10.2144/000112908

Cited by 23 publications

(19 citation statements)

References 6 publications

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“…Flow cytometry is a classical method to detect cell apoptosis with high sensitivity and can be used for simultaneous cell-cycle analysis [ 26 ]. However, a general laboratory may not be equipped with a flow cytometer, and they are costly to run.…”

Section: Discussionmentioning

confidence: 99%

Dual AO/EB Staining to Detect Apoptosis in Osteosarcoma Cells Compared with Flow Cytometry

2015

Med Sci Monit Basic Res

543

128

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BackgroundThe 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.Material/MethodWe 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.ResultNormal 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).ConclusionsOur 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.

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Section: Discussionmentioning

confidence: 99%

Dual AO/EB Staining to Detect Apoptosis in Osteosarcoma Cells Compared with Flow Cytometry

2015

Med Sci Monit Basic Res

543

128

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“…Flow cytometry of TCTF-stained cells was done according to Meyer et al [28] using a LSRFortessa flow cytometer (BD biosciences). Alternatively cells were stained with propidium iodide (5 µg/ml) and a FITC-conjugated Annexin V antibody (BD Biosciences) according to manufacturer instructions.…”

Section: Methodsmentioning

confidence: 99%

Meningococcal Outer Membrane Protein NhhA Triggers Apoptosis in Macrophages

et al. 2012

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Phagocytotic cells play a fundamental role in the defense against bacterial pathogens. One mechanism whereby bacteria evade phagocytosis is to produce factors that trigger apoptosis. Here we identify for the first time a meningococcal protein capable of inducing macrophage apoptosis. The conserved meningococcal outer membrane protein NhhA (Neisseria hia/hsf homologue A, also known as Hsf) mediates bacterial adhesion and interacts with extracellular matrix components heparan sulphate and laminin. Meningococci lacking NhhA fail to colonise nasal mucosa in a mouse model of meningococcal disease. We found that exposure of macrophages to NhhA resulted in a highly increased rate of apoptosis that proceeded through caspase activation. Exposure of macrophages to NhhA also led to iNOS induction and nitric oxide production. However, neither nitric oxide production nor TNF-α signaling was found to be a prerequisite for NhhA-induced apoptosis. Macrophages exposed to wildtype NhhA-expressing meningococci were also found to undergo apoptosis whereas NhhA-deficient meningococci had a markedly decreased capacity to induce macrophage apoptosis. These data provide new insights on the role of NhhA in meningococcal disease. NhhA-induced macrophage apoptosis could be a mechanism whereby meningococci evade immunoregulatory and phagocytotic actions of macrophages.

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“…This assay helps with detection and quantification of apoptosis. The APOPercentage assay was performed following a literature procedure [47]. In the pro-apoptotic activity of the complexes (Fig.…”

Section: Figmentioning

confidence: 99%

Bis(ferrocenylimine)palladium(II) and platinum(II) complexes: Synthesis, molecular structures and evaluation as antitumor agents

Motswainyana¹,

Onani²,

Madiehe³

2012

Polyhedron

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A Low-Cost Flow Cytometric Assay for the Detection and Quantification of Apoptosis Using an Anionic Halogenated Fluorescein Dye

Cited by 23 publications

References 6 publications

Dual AO/EB Staining to Detect Apoptosis in Osteosarcoma Cells Compared with Flow Cytometry

Dual AO/EB Staining to Detect Apoptosis in Osteosarcoma Cells Compared with Flow Cytometry

Meningococcal Outer Membrane Protein NhhA Triggers Apoptosis in Macrophages

Bis(ferrocenylimine)palladium(II) and platinum(II) complexes: Synthesis, molecular structures and evaluation as antitumor agents

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