7-Aminoactinomycin D for apoptosis staining in flow cytometry

Zembruski, Nadine Cécile Luise; Stache, Vanessa; Haefeli, Walter E.; Weiß, Johanna

doi:10.1016/j.ab.2012.07.005

Cited by 103 publications

(77 citation statements)

References 13 publications

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“…To measure apoptotic cells, 10 μg/ml 7-amino actinomycin D (7-AAD, Life Technologies) (39) was added to 0.2-0.5 × 10 6 cells collected in 100 μl FACS-buffer (1 × PBS, 2 mM EDTA) and incubated for 15 min at 4°C before determining the percentages of 7-AAD + cells. Here, after gating on viable cells in the SSC-A/FSC-A scatter-plot, GFP + cells were gated and checked for the percentage of 7-AAD + GFP + cells.…”

Section: Methodsmentioning

confidence: 99%

The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation

et al. 2016

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RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3′UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3′UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response.

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Section: Methodsmentioning

confidence: 99%

The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation

et al. 2016

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“…Cells were stained with an antibody against CD144 to identify endothelial cells, and the viability of the endothelial cells was determined by flow cytometry based on 7-AAD staining. 39 Endothelial cell death was expressed as the percentage of endothelial cells that stained positively with dye. Approximately 10,000 cells were counted in each sample.…”

Section: Methodsmentioning

confidence: 99%

Photoreceptor Cells Influence Retinal Vascular Degeneration in Mouse Models of Retinal Degeneration and Diabetes

Liu

Tang

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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PurposeLoss of photoreceptor cells is associated with retinal vascular degeneration in retinitis pigmentosa, whereas the presence of photoreceptor cells is implicated in vascular degeneration in diabetic retinopathy. To investigate how both the absence and presence of photoreceptors could damage the retinal vasculature, we compared two mouse models of photoreceptor degeneration (opsin−/− and RhoP23H/P23H ) and control C57Bl/5J mice, each with and without diabetes.MethodsRetinal thickness, superoxide, expression of inflammatory proteins, ERG and optokinetic responses, leukocyte cytotoxicity, and capillary degeneration were evaluated at 1 to 10 months of age using published methods.ResultsRetinal photoreceptor cells degenerated completely in the opsin mutants by 2 to 4 months of age, and visual function subsided correspondingly. Retinal capillary degeneration was substantial while photoreceptors were still present, but slowed after the photoreceptors degenerated. Diabetes did not further exacerbate capillary degeneration in these models of photoreceptor degeneration, but did cause capillary degeneration in wild-type animals. Photoreceptor cells, however, did not degenerate in wild-type diabetic mice, presumably because the stress responses in these cells were less than in the opsin mutants. Retinal superoxide and leukocyte damage to retinal endothelium contributed to the degeneration of retinal capillaries in diabetes, and leukocyte-mediated damage was increased in both opsin mutants during photoreceptor cell degeneration.ConclusionsPhotoreceptor cells affect the integrity of the retinal microvasculature. Deterioration of retinal capillaries in opsin mutants was appreciable while photoreceptor cells were present and stressed, but was less after photoreceptors degenerated. This finding proves relevant to diabetes, where persistent stress in photoreceptors likewise contributes to capillary degeneration.

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“…To assess the cytocompatibility of NPs, dendritic cells were incubated with empty and OVA NPs as well as dual labeled NPs for 16 h at 37 C. Dead cells were quantified by using 7-AAD, a DNAbinding fluorescent dye which stains cells that have lost membrane integrity [78]. Dendritic cells incubated with LPS and soluble OVA corresponding to the amounts of OVA in the loaded nanoparticles were used as controls.…”

Section: Cytocompatibility Of Empty and Ova-loaded Npsmentioning

confidence: 99%

Near-infrared labeled, ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine: In vivo tracking and evaluation of antigen-specific CD8 + T cell immune response

et al. 2015

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7-Aminoactinomycin D for apoptosis staining in flow cytometry

Cited by 103 publications

References 13 publications

The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation

The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation

Photoreceptor Cells Influence Retinal Vascular Degeneration in Mouse Models of Retinal Degeneration and Diabetes

Near-infrared labeled, ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine: In vivo tracking and evaluation of antigen-specific CD8 + T cell immune response

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