Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis.
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ABSTRACT: Alcohol impairs the host immune system, rendering hosts more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here we showed that S. pneumoniae type 2 D39 is ethanol tolerant, and that alcohol up-regulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, pro-inflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE in the ethanol condition conferred an increase of Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than ΔadhE during the ethanol stress condition, and alcoholic mice were more susceptible to infection with the D39 wild-type bacteria than the ΔadhE. Taken together, these data indicate that AdhE increases Ply in the ethanol stress condition, thus potentiating pneumococcal virulence.Infection and Immunity 01/2015; · 4.16 Impact Factor
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ABSTRACT: Vitamin A and its metabolic derivative, retinoic acid (RA) are essential biomolecules for normal development regulation and regeneration in different organs. Too much and deficiency of vitamin A can be toxic for fetuses lead to birth defects or is associated with many congenital malformations. It has been known that RA alone or in combination with other morphogens promotes the developmental program such as neural differentiation and patterning. Here, we discussed RA role in the neural patterning of the embryonic stem cells and its function in promoting the neural differentiation in neural plate of the developing embryo through attenuating the fibroblast growth factor (FGF) signaling. By using different techniques, we also argued the opposite function of RA in inducing apoptosis in the human umbilical cord-derived mesenchymal stem cells (hUCMSCs) shown by upregulating the caspase expression. Finally, we discussed that some biological parameters including cell density and passage appeared to be involved in this cytotoxicity responseStem Cell and Translational Investigation. 02/2015; 2(1):1-3.
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ABSTRACT: Carrageenan is a linear sulphated polysaccharide extracted from red seaweed of the Rhodophyceae family. It has broad spectrum of applications in biomedical and biopharmaceutical field. In this study, we determined the cytotoxicity of degraded and undegraded carrageenan in human intestine (Caco-2; cancer and FHs 74 Int; normal) and liver (HepG2; cancer and Fa2N-4; normal) cell lines. Food grade k-carrageenan (FGKC), dried sheet k-carrageenan (DKC), commercial grade k-carrageenan (CGKC), food grade i-carrageenan (FGIC) and commercial grade i-carrageenan (CGIC) were dissolved in hydrochloric acid and water to prepare degraded and undegraded carrageenan, respectively. Carrageenan at the concentration range of 62.5 - 2000.0 mug mL-1 was used in the study. MTT assay was used to determine the cell viability while the mode of cell death was determined by May-Grunwald Giemsa (MGG) staining, acridine orange-ethidium bromide (AO/EtBr) staining, agarose gel electrophoresis and gene expression analysis. Degraded FGKC, DKC and CGKC showed IC50 in 24, 48 and 72 hours treated Caco-2, FHs 74 Int, HepG2 and Fa2N-4 cell lines as tested by MTT assay. Degraded FGIC and CGIC only showed its toxicity in Fa2N-4 cells. The characteristics of apoptosis were demonstrated in degraded k-carrageenan treated Caco-2, FHs 74 Int, HepG2 and Fa2N-4 cells after MGG staining. When Caco-2 and HepG2 cells were undergone AO/EtBr staining, chromatin condensation and nuclear fragmentation were clearly seen under the microscope. However, DNA ladder was only found in HepG2 cells after gel electrophoresis analysis. Degraded k-carrageenan also inactivated PCNA, Ki-67 and survivin gene in HepG2. On the other hand, undegraded FGKC, DKC, CGKC, FGIC and CGIC treated cells showed no cytotoxic effect after analyzed by the same analyses as in degraded carrageenan. Degraded k-carrageenan inhibited cell proliferation in Caco-2, FHs 74 Int, HepG2 and Fa2N-4 cell lines and the anti-proliferative effect was related to apoptosis together with inactivation of cell proliferating genes as determined by morphological observation and molecular analysis. However, no cytotoxic effect was found in undegraded carrageenan towards normal and cancer intestine and liver cell lines.BMC Complementary and Alternative Medicine 12/2014; 14(1):508. · 1.88 Impact Factor
Cold Spring Harbor Protocols
Cold Spring Harbor Protocols
Cold Spring Harb Protoc 2006. 2006: pdb.prot4493-
Acridine Orange/Ethidium Bromide (AO/EB)
Staining to Detect Apoptosis
This protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector et
al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This
three-volume set is now out of print; however, some of the microscopy methods were
republished in Basic Methods in Microscopy, by David L. Spector and Robert D.
Shailaja Kasibhatla, , Gustavo P. Amarante-Mendes
Thomas Brunner, , Ella Bossy-Wetzel
Gustavo P. Amarante-Mendes, , Deborah Finucane
Ella Bossy-Wetzel and
Deborah Finucane, ,
and Douglas R. GreenDouglas R. Green
Acridine orange/ethidium bromide (AO/EB) staining is used to visualize nuclear
changes and apoptotic body formation that are characteristic of apoptosis. Cells
are viewed under a fluorescence microscope and counted to quantify apoptosis.
Fluorescence microscope with fluorescein filter and 60X objective
Slides and coverslips
1. Incubate 25 µl of cell suspension (0.5 × 106 to 2.0 × 106 cells/ml)
with 1 µl of AO/EB solution. Mix gently. Each sample should be mixed
just prior to microscopy and quantification. Samples must be
2. Place 10 µl of cell suspension onto a microscopic slide, cover with a
glass coverslip, and examine at least 300 cells in a fluorescence
microscope using a fluorescein filter and a 60X objective. (Higher or
lower magnification may be desired depending on cell type. Nuclear
morphology should be discernible.)
Acridine orange is a vital dye and will stain both live and dead cells.
Ethidium bromide will stain only cells that have lost membrane
integrity. Live cells will appear uniformly green. Early apoptotic cells
will stain green and contain bright green dots in the nuclei as a
consequence of chromatin condensation and nuclear fragmentation.
Late apoptotic cells will also incorporate ethidium bromide and
therefore stain orange, but, in contrast to necrotic cells, the late
apoptotic cells will show condensed and often fragmented nuclei.
Necrotic cells stain orange, but have a nuclear morphology
resembling that of viable cells, with no condensed chromatin.