Use of the MTT Assay in Adult Ventricular Cardiomyocytes to Assess Viability: Effects of Adenosine and Potassium on Cellular Survival

Gómez, Luis Alberto; Alekseev, Alexey E.; Aleksandrova, Luba A.; Brady, Peter A.; Terzic, André

doi:10.1006/jmcc.1996.0363

Cited by 80 publications

(50 citation statements)

References 4 publications

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“…Cell viability assay [25] Cell viability was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide (MTT) assay. The HUVECs were seeded in 96-well plates at 1×10 5 cells/well.…”

Section: Drug Treatmentmentioning

confidence: 99%

The protective effects of Polygonum multiflorum stilbeneglycoside preconditioning in an ischemia/reperfusion model of HUVECs

et al. 2010

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Aim: To investigate the protective effects of preconditioning human umbilical vein endothelial cells (HUVECs) with Polygonum multiflorum stilbeneglycoside (PMS) under anoxia/reoxygenation (A/R), and the mechanism of protection. Methods: Prior to A/R, HUVECs were incubated with PMS (0.6×10 , or 2.4×10 -11 mol/L) for 3 h. Cell injury was subsequently evaluated by measuring cell viability with an MTT assay and lactate dehydrogenase (LDH) release, whereas lipid peroxidation was assayed by measuring malondialdehyde (MDA) content. Antioxidant capacity was quantified by superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. Nitric oxide (NO) production was determined by nitrite accumulation. Endothelial NO synthase (eNOS) and inducible NOS (iNOS) protein expression was detected by Western blotting. Guanylate cyclase activity and cyclic GMP (cGMP) activity were assessed by an enzyme immunoassay kit. Results: PMS incubation attenuated A/R-induced injury in a concentration-dependent manner, as evidenced by a decrease in LDH activity and an increase in cell viability. PMS exerted its protective effect by inhibiting the A/R-mediated elevation of MDA content, as well as by promoting the recovery of SOD and GSH-Px activities. Additionally, PMS incubation enhanced NO and cGMP formation by increasing iNOS expression and guanylate cyclase activity. The protective effects of PMS were markedly attenuated by NOS inhibitor L-NAME, soluble guanylate cyclase inhibitor ODQ or PKG inhibitor KT5823.Conclusion: PMS preincubation resulted in the enhancement of antioxidant activity and anti-lipid peroxidation. The NO/cGMP/cGMPdependent protein kinase (PKG) signaling pathway was involved in the effect of PMS on HUVECs.

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Section: Drug Treatmentmentioning

confidence: 99%

The protective effects of Polygonum multiflorum stilbeneglycoside preconditioning in an ischemia/reperfusion model of HUVECs

et al. 2010

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“…Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay (Gomez et al, 1997). BAEC were seeded into 96-well plates at a density of 1·10 5 cells/well in DMEM-F12 and cultivated overnight until cell attachment.…”

Section: Methodsmentioning

confidence: 99%

Monomethylarsonous Acid Induced Cytotoxicity and Endothelial Nitric Oxide Synthase Phosphorylation in Endothelial Cells

Sun

et al. 2007

Bull Environ Contam Toxicol

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Chronic arsenic poisoning is reported to be associated with peripheral and cardiovascular disease, arteriosclerosis, Raynaud's syndrome, hypertension, and Blackfoot disease. Monomethylarsonous acid (MMA III ) is a reactive metabolite of inorganic arsenic and a potent inhibitor of endothelial nitric oxide synthase (eNOS). Arsenic is also reported to phosphorylate eNOS in cultured keratinocyte and Human T cell leukemia Jurkat cells, respectively. In the present study, we examined the cytotoxicity and eNOS phosphorylation by MMA III exposure in cultured bovine aortic endothelial cells (BAEC). Results showed that MMA III is more toxic than arsenite in BAEC cells. The IC 50 values for MMA III and arsenite were determined to be approximately 1.7 and 24.1 lmol/L, respectively. Exposure of BAEC to MMA III (0.75 lmol/L) caused a significant eNOS phosphorylation 15 min after MMA III exposure. However, a complex of MMA III with dithiothreitol (DTT) that lacks the reactivity with vicinal thiols unaffected eNOS phosphorylation. The present study shows that MMA III generated during biomethylation of arsenic is highly toxic in BAEC. Our study also suggests that MMA III could induce the eNOS phosphorylation through modification to cellular thiols of the eNOS enzyme. And the initial up-regulation of eNOS phosphorylation by MMA III seems to be an adaptive response against disruption of eNOS bioactivity during arsenic exposure.

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“…La viabilidad celular de cardiomiocitos aislados individuales se evaluó mediante el recuento de las células que excluían el Tripán azul (0,25%) (Gomez, 1997). Las células individuales se clasificaron en uno de los siguientes tres grupos fenotípicos: (1) Células en forma de bastón con clara forma alargada con estriado de sarcómeras definidas, membrana celular lisa y regular, que excluían el Tripán azul; (2) Células de forma redonda sin estrías claras y contractura aparente, que incluían el azul tripán, y (3) Células de forma rectangular, con estrías y superficie regular, que excluyeron el azul tripán.…”

Section: Morfología Y Exclusión Del Tripán Azulunclassified

“…La función de la membrana celular se altera, lo cual lleva a liberación de potasio, captación de sodio y cambios en las concentraciones de calcio libre por los cardiomiocitos. La severidad y duración de la hipoxia determinan si los cambios celulares son transitorios o permanentes, en cuyo caso la célula muere (Buerke et al, 1994;Gomez et al, 1997).…”

Section: Introductionunclassified

“…Una de las áreas de mayor investigación en el área de la cardiología molecular está orientada a definir los determinantes moleculares involucrados en la hipoxia cardiaca, en la supervivencia y funcionalidad de los cardiomiocitos y en los mecanismos moleculares de un fenómeno de cardioprotección conservado, que protege al corazón de la hipoxia y estrés metabólico y físico, conocido como preacondicionamiento isquémico cardiaco (PCI) (Murray, 1986;Gomez, 1997;Marber, 2000;Amstrong, 2004, Gross, 2013. No obstante, la mayoría de las investigaciones en PCI se han realizado en corazones completos y pocas en células cardiacas aisladas.…”

Section: Introductionunclassified

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Preacondicionamiento isquémico en cardiomiocitos ventriculares aislados. Identificación y expresión de algunos microRNAs asociados

Gómez-Grosso

2014

Rev. Acad. Colomb. Cienc. Ex. Fis. Nat.

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RESUMENGómez-Grosso L.A.: Preacondicionamiento isquémico en cardiomiocitos ventriculares aislados. Identificación y expresión de algunos microRNAs asociados. Rev. Acad. Colomb. Cienc. 37 (145): [433][434][435][436][437][438][439][440][441][442][443][444][445][446][447] 2013. ISSN 0370-3908.El objetivo de este estudio fue evaluar el preacondicionamiento isquémico, PCI, en cardiomiocitos ventriculares de cobayo adultos individuales y evaluar el perfil de expresión diferencial de microRNAs en estas células. Una supervisión de la morfología, de la actividad metabólica de las células cardíacas individuales aisladas en tiempo real, permite una evaluación de los cambios en la viabilidad de los cardiomiocitos y proporciona evidencia experimental de que el PCI tiene acción citoprotectora directa mediante la promoción de la supervivencia in vitro. La protección cardiaca por PCI asociada con la expresión diferencial de microRNAs específicos en cardiomicitos adultos aislados sugiere que algunos miRNAs están involucrados en lesión y cardioprotección.Palabras clave: cardioprotección, preacondicionamiento, ensayo de MTT, tiempo real, cardiomiocitos, viabilidad, isquemia, Dinitrofenol, Adenosina. ABSTRACTThe aim of this study was to evaluate the ischemic preconditioning, PCI, in single quiescent adult guinea-pig ventricular myocytes and assess the differential expression of microRNAs in these cells. Monitoring of the morphology of the metabolic activity of individual cardiac cells isolated in real time, allows evaluation of changes in the viability of the cardiomyocytes and provides experimental evidence that PCI has direct cytoprotective action by promoting survival in vitro. PCI cardiac protection associated with the differential expression of specific microRNAs in isolated adult cardiomyocytes suggests that miRNAs are involved in some injury and cardioprotection.

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Use of the MTT Assay in Adult Ventricular Cardiomyocytes to Assess Viability: Effects of Adenosine and Potassium on Cellular Survival

Cited by 80 publications

References 4 publications

The protective effects of Polygonum multiflorum stilbeneglycoside preconditioning in an ischemia/reperfusion model of HUVECs

The protective effects of Polygonum multiflorum stilbeneglycoside preconditioning in an ischemia/reperfusion model of HUVECs

Monomethylarsonous Acid Induced Cytotoxicity and Endothelial Nitric Oxide Synthase Phosphorylation in Endothelial Cells

Preacondicionamiento isquémico en cardiomiocitos ventriculares aislados. Identificación y expresión de algunos microRNAs asociados

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