An improved MTT assay using the electron-coupling agent menadione

Garn, Holger; Krause, Heidi; Enzmann, Volker; Dröβler, Karl

doi:10.1016/0022-1759(94)90062-0

Cited by 58 publications

(32 citation statements)

References 5 publications

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“…It is based on the conversion of the yellow tetrazolium salt MTT to the blue formazan derivative by mitochondria enzymes in viable cells. 16 We used an improved version of the assay. 17 Our MTT assays confirmed the results of the Hoechst33342 staining.…”

Section: Resultsmentioning

confidence: 99%

“…The second was the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, which was carried out according to an established protocol. 16 Phospho-p38 MAPK Assay p38 MAPK activity was measured using a nonradioactive immobilized phospho-p38 MAPK assay kit purchased from Cell Signaling Technology, Inc. Manufacturer's instructions were followed to carry out the assay.…”

Section: Cell Death Assaysmentioning

confidence: 99%

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Protection Against Oxidized Low-Density Lipoprotein–Induced Vascular Endothelial Cell Death by Integrin-Linked Kinase

Zhang

Hu²,

Li³

2001

Circulation

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Background-Integrin-linked kinase (ILK) is a protein that plays important roles in extracellular matrix-mediated signaling. It has been shown that ILK is expressed preferentially in cardiac and skeletal muscles. Evidence points to its role as an upstream regulator of protein kinase B, a critical player in apoptosis. Because oxidized LDL (oxLDL) is thought to promote atherogenesis by causing the apoptosis of endothelial cells, we investigated the potential roles that ILK may play in oxLDL-induced apoptosis in vascular endothelial cells. Methods and Results-Transcriptional and translational levels of ILK were investigated with reverse-transcriptase polymerase chain reaction and Western analysis. oxLDL treatment induced both the transcription and the translation of the ILK gene in endothelial cells. A recombinant adenovirus vector encoding the ILK gene was constructed to investigate its potential role in oxLDL-induced apoptosis in human umbilical vein endothelial cells and mouse lymphoid vein endothelial cells transformed by simian virus 40. In both types of cells, overexpression of the ILK gene significantly prevented oxLDL-induced apoptosis or cell death, as evaluated by 2 independent assay methods. Furthermore, we showed that ILK could inhibit oxLDL-induced upregulation of the kinase activity of p38 mitogen-activated protein kinase, which is often associated with stress-induced pro-apoptotic signal transduction. Finally, examination of other factors, such as bcl-2, bcl-xl, caspase 3, and caspase 9, demonstrated significant changes that were correlated with oxLDL treatment and ILK overexpression. Key Words: oxygen Ⅲ lipoproteins Ⅲ endothelium Ⅲ apoptosis Ⅲ kinases O xidized LDL (oxLDL) has been implicated in atherosclerosis in both epidemiological and laboratory studies. 1,2 The precise role that oxLDL plays in atherogenesis is unknown. There is, however, increasing evidence to suggest that oxLDL-induced injury of the vascular wall is critical because it may be the initiating event that leads to the inflammatory responses now recognized to be the main cause of atherosclerosis. How does oxLDL induce injury to the vascular wall? Many recent studies suggest that oxLDLinduced apoptosis in the vascular smooth muscle cells or vascular endothelial cells may be the key. In support of this theory, several studies have demonstrated the presence of apoptosis in human and experimental atherosclerotic plaques. 3,4 Tissue culture experiments have indicated that oxLDL exposure leads to extensive apoptosis in vascular smooth muscle cells, 5 human coronary cells, 6 and human umbilical venous endothelial cells (HUVECs). 7,8 It has been further shown that the p38 mitogen-activated protein kinase (MAPK) is a downstream effector of the oxLDLinduced apoptotic process. 5 Little is known, however, about the upstream molecular mechanisms that regulate this process. Conclusion-ILKIn our efforts to identify novel signal-transduction pathways further upstream, we came across integrin-linked kinase (ILK) by virtue of its upregulation in oxLD...

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Section: Resultsmentioning

confidence: 99%

Section: Cell Death Assaysmentioning

confidence: 99%

Protection Against Oxidized Low-Density Lipoprotein–Induced Vascular Endothelial Cell Death by Integrin-Linked Kinase

Zhang

Hu²,

Li³

2001

Circulation

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“…In order to determine the impact of F. nucleatum on endothelial cell proliferation, we used an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (21)(22)(23). Endothelial cells were seeded at 1 ϫ 10 4 cells/well in 96-well plates and incubated for 24 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Endothelial Cell Response to Fusobacterium nucleatum

Mendes

Nguyen²,

Stephens³

et al. 2016

Infect Immun

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c Vascular response is an essential aspect of an effective immune response to periodontal disease pathogens, as new blood vessel formation contributes to wound healing and inflammation. Gaining a greater understanding of the factors that affect vascular response may then contribute to future breakthroughs in dental medicine. In this study, we have characterized the endothelial cell response to the common bacterium Fusobacterium nucleatum, an important bridging species that facilitates the activity of late colonizers of the dental biofilm. Endothelial cells were infected with Fusobacterium nucleatum (strain 25586) for periods of 4, 12, 24, or 48 h. Cell proliferation and tube formation were analyzed, and expression of adhesion molecules (CD31 and CD34) and vascular endothelial growth factor (VEGF) receptors 1 and 2 was measured by fluorescenceactivated cell sorter (FACS) analysis. Data indicate that F. nucleatum impaired endothelial cell proliferation and tube formation. The findings suggest that the modified endothelial cell response acts as a mechanism promoting the pathogenic progression of periodontal diseases and may potentially suggest the involvement of periodontopathogens in systemic diseases associated with periodontal inflammation.

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“…The formazan product was dissolved in 10% sodium dodecyl sulfate 100 µL containing hydrochloric acid 15 mM. 25 Color intensity was measured using an absorbance microplate reader (ELx800™, BioTek, Winooski, VT) at test and reference wavelengths of 570 nm.…”

Section: Gel Retardation Analysis Of Lipoplexesmentioning

confidence: 99%

Preparation, characterization, and efficient transfection of cationic liposomes and nanomagnetic cationic liposomes

Samadikhah¹,

Majidi²,

Nikkhah³

et al. 2011

IJN

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Purpose: Cationic liposomes (CLs) are composed of phospholipid bilayers. One of the most important applications of these particles is in drug and gene delivery. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs has some problems, including low transfection efficiency in vivo. The aim of this study was to develop novel CLs containing magnetite to overcome the deficiencies. Materials and methods: CLs and magnetic cationic liposomes (MCLs) were prepared using the freeze-dried empty liposome method. Luciferase-harboring vectors (pGL3) were transferred into liposomes and the transfection efficiencies were determined by luciferase assay. Firefly luciferase is one of most popular reporter genes often used to measure the efficiency of gene transfer in vivo and in vitro. Different formulations of liposomes have been used for delivery of different kinds of gene reporters. Lipoplex (liposome-plasmid DNA complexes) formation was monitored by gel retardation assay. Size and charge of lipoplexes were determined using particle size analysis. Chinese hamster ovary cells were transfected by lipoplexes (liposome-pGL3); transfection efficiency and gene expression level was evaluated by luciferase assay. Results: High transfection efficiency of plasmid by CLs and novel nanomagnetic CLs was achieved. Moreover, lipoplexes showed less cytotoxicity than polyethyleneimine and Lipofectamine ™ . Conclusion: Novel liposome compositions (1,2-dipalmitoyl-sn-glycero-3-phosphocholine [DPPC]/dioctadecyldimethylammonium bromide [DOAB] and DPPC/cholesterol/DOAB) with high transfection efficiency can be useful in gene delivery in vitro. MCLs can also be used for targeted gene delivery, due to magnetic characteristic for conduction of genes or drugs to target organs.

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An improved MTT assay using the electron-coupling agent menadione

Cited by 58 publications

References 5 publications

Protection Against Oxidized Low-Density Lipoprotein–Induced Vascular Endothelial Cell Death by Integrin-Linked Kinase

Protection Against Oxidized Low-Density Lipoprotein–Induced Vascular Endothelial Cell Death by Integrin-Linked Kinase

Endothelial Cell Response to Fusobacterium nucleatum

Preparation, characterization, and efficient transfection of cationic liposomes and nanomagnetic cationic liposomes

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