Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide
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Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.Keywords:
Propidium iodide
Acridine orange
Ethidium bromide
Trypan blue
Annexin A5
Agarose gel electrophoresis
Viability assay
Acridine orange
Ethidium bromide
Metachromasia
Acridine
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The plasmid eliminating abilities of acridine orange, ethidium bromide and sodium dodecyl sulfate were investigated on multi drug resistant Escherichia coli from urinary tract infection specimens. Three different concentrations of each curing agent (Et-Br, SDS and AO) were used. The frequencies of cured cells were 5.55 % (with 50 μg/ml) and 11.76 % (with 75 μg/ml) for acridine orange, 14.29 % (with 100 μg/ml), 21.05 % (with 100 μg/ ml), 17.65 % (with 125 μg/ml) for ethidium bromide and 7.4 % (with 10 % w/v) & 6.67 % (with 10 % w/v) for sodium dodecyl sulfate. However, no cured cells were obtained from 100 μg/ml acridine orange, 75 μg/ml ethidium bromide and 8 and 12 % SDS. Analysis of profiles of wild type and plasmid cured strains by electrophoresis yielded bands of varying sizes for wild type cells, but none were obtained for Et-Br cured cells. Acridine orange treated cells could eliminate only plasmids of 2.7 MDa and another smaller than 2 MDa. Key Words: Plasmid curing; Escherichia coli; Ethidium Bromide; Sodium Dodecyl Sulfate; Acridine Orange. DOI: http://dx.doi.org/10.3329/bjm.v27i1.9165 BJM 2010; 27(1): 28-31
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The interaction of several aromatic cationic dyes such as, ethidium bromide (EB), methylene blue (MB), acridine orange (AO), and Hoechst 33258 with calf-thymus DNA and poly(A)-poly(U) duplex was investigated. The different induced extrinsic Cotton effects (greater than 300 nm) were observed for DNA- and RNA-dye complexes. The binding properties of these complexes were examined by UV, CD, and NMR spectroscopies.
Ethidium bromide
Acridine orange
Cationic polymerization
Methylene blue
Duplex (building)
Proton NMR
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Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.
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Acridine orange enhances the uptake of [3H]actinomycin D in disrupted and intact human lymphocytes, as measured by liquid scintillation and autoradiography. Proflavine, quinacrine, chloroquine, and ethidium bromide are not effective. In mice, acridine orange increases the capacity of actinomycin to reduce spleen weight. Type II acridine binding to DNA may be a prerequisite for actinomycin enhancement.
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Ethidium bromide
Proflavine
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Dactinomycin
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Acridine orange
Ethidium bromide
Fluorescent staining
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Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4′,6‐diamidino‐2‐phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.
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A cytochemical method of chromatin study by means of nuclear staining with ethidium (bromide) is described. Dependence of stain binding by chromatin on ethidium concentration, ionic composition and buffer pH value has been analyzed. It is suggested that cells be stained in 2.10(-5) M solution of ethidium in 0.1 M tris-HCl buffer at pH 8.0 during 30 min. The fluorescence of nuclei stained with ethidium under conditions described is shown to reflect changes in physico-chemical properties of chromatin taking place in the course of its chemical modification and physiological activation in regenerating liver. The use of ethidium for chromatin cytochemistry allows to study chromatin properties in wide ranges of pH. Some other advantages of the method suggested over the commonly used method of acridine orange staining are discussed.
Ethidium bromide
Acridine orange
Cytochemistry
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