A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture

Mironova, Elena; Evstratova, Alesya; Антонов, С. М.

doi:10.1016/j.jneumeth.2007.02.010

Cited by 89 publications

(59 citation statements)

References 33 publications

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“…The procedure of culture preparation from embryos was previously described (Antonov and Johnson, 1996;Mironova et al, 2007). All procedures using animals were in accordance with recommendations of the Federation for Laboratory Animal Science Associations and approved by the local Institutional Animal Care and Use Committees.…”

Section: Methodsmentioning

confidence: 99%

“…Fetuses were removed, and their cerebral cortices were isolated, enzymatically dissociated, and used to prepare primary neuronal cultures. Cells were used for experiments after 10-15 days in culture (Mironova et al, 2007;Han and Stevens, 2009). Cells were grown in Neurobasal culture medium supplemented with B-27 (Gibco-Invitrogen, Paisley, UK) on glass coverslips coated with poly-D-lysine.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation

Сибаров

Абушик

Poguzhelskaya

et al. 2015

J Pharmacol Exp Ther

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation

Сибаров

Абушик

Poguzhelskaya

et al. 2015

J Pharmacol Exp Ther

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“…Cell cultures were prepared as described previously (Antonov et al, 1998;Mironova et al, 2007). All procedures using animals were in accordance with recommendations of the Federation for Laboratory Animal Science Associations and approved by the local Institutional Animal Care and Use Committees.…”

Section: Methodsmentioning

confidence: 99%

Na⁺,K⁺-ATPase Functionally Interacts with the Plasma Membrane Na⁺,Ca²⁺ Exchanger to Prevent Ca²⁺ Overload and Neuronal Apoptosis in Excitotoxic Stress

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Bolshakov

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et al. 2012

J Pharmacol Exp Ther

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Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca 2ϩ imaging analysis, we report that ouabain, a specific ligand of the Na ϩ ,K ϩ -ATPase cardiac glycoside binding site, can prevent glutamate receptor agonistinduced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 M N-methyl-Daspartate (NMDA) or kainate caused apoptosis in ϳ50% of neurons. These effects were accompanied by a significant decrease in the number of neurons that were immunopositive for the antiapoptotic peptide Bcl-2. Apoptotic injury was completely prevented when the agonists were applied together with 0.1 or 1 nM ouabain, resulting in a greater survival of neurons, and the percentage of neurons expressing Bcl-2 remained similar to those obtained without agonist treatments. In addition, subnanomolar concentrations of ouabain prevented the increase of spontaneous excitatory postsynaptic current's frequency and the intracellular Ca 2ϩ overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid or inhibition of the plasma membrane Na ϩ ,Ca 2ϩ -exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabain's effects on NMDA-or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca 2ϩ extrusion from neurons via the Na ϩ ,Ca 2ϩ exchanger. Because intracellular Ca 2ϩ accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca 2ϩ extrusion abolishes its development. These antiapoptotic effects are independent of Na ϩ ,K ϩ -ATPase ion transport function and are initiated by concentrations of ouabain that are within the range of an endogenous analog, suggesting a novel functional role for Na ϩ ,K ϩ -ATPase in neuroprotection.

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“…The acr idine orange/ethidium bromide vital staining technique was used to determine the ratio of living to apoptotic cells, as previously described (11). The cells were incubated for 3 days with Pin1 siRNA or non-targeting siRNA in order to observe knockdown at the protein level.…”

Section: Methodsmentioning

confidence: 99%

Knockdown of Pin1 leads to reduced angiogenic potential and tumorigenicity in glioblastoma cells

et al. 2015

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Abstract. Glioblastoma is the most common and most aggressive type of primary brain tumor. Current approaches in the treatment of glioblastoma are not effective enough to increase patient survival or prevent recurrence following surgery. Consequently, the search for potential drug targets is ongoing. Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1), an isomerase that is overexpressed in various tumors, has become an attractive molecule in cancer research. Pin1 has been reported to regulate proteins involved in essential cellular pathways that mediate cell proliferation, cell cycle progression, differentiation and apoptosis, by altering their stability and function. The results of the present study revealed that knockdown of Pin1 in glioblastoma cells using RNA interference or the selective Pin1 inhibitor, juglone, suppressed the tumorigenic features by reducing cell growth, migration and angiogenic potential. Furthermore, knockdown of Pin1 decreased the levels of vascular endothelial growth factor and matrix metallopeptidase 9, and also triggered apoptosis. Due to the fundamental roles of Pin1 in promoting tumorigenesis, Pin1 inhibitory molecules, including juglone, or alternative synthetic derivatives hold potential for the development of clinical countermeasures against glioblastoma.

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A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture

Cited by 89 publications

References 33 publications

Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation

Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation

Na⁺,K⁺-ATPase Functionally Interacts with the Plasma Membrane Na⁺,Ca²⁺ Exchanger to Prevent Ca²⁺ Overload and Neuronal Apoptosis in Excitotoxic Stress

Knockdown of Pin1 leads to reduced angiogenic potential and tumorigenicity in glioblastoma cells

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A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture

Cited by 89 publications

References 33 publications

Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation

Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation

Na+,K+-ATPase Functionally Interacts with the Plasma Membrane Na+,Ca2+ Exchanger to Prevent Ca2+ Overload and Neuronal Apoptosis in Excitotoxic Stress

Knockdown of Pin1 leads to reduced angiogenic potential and tumorigenicity in glioblastoma cells

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Na⁺,K⁺-ATPase Functionally Interacts with the Plasma Membrane Na⁺,Ca²⁺ Exchanger to Prevent Ca²⁺ Overload and Neuronal Apoptosis in Excitotoxic Stress