Quantification of neuron survival in monolayer cultures using an enzyme-linked immunosorbent assay approach, rather than by cell counting

Brooke, Sheila M.; Bliss, Tonya; Franklin, Laura; Sapolsky, Robert M.

doi:10.1016/s0304-3940(99)00315-8

Cited by 46 publications

(32 citation statements)

References 15 publications

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“…The 2,3Ј-azino-bis(ethylbenzothiazoline-6-sulfonic acid) (ABTS)/enzyme-linked immunosorbent assay method used for quantification of cell death is a fast, reliable, and objective procedure (Brooke et al, 1999). Cells were fixed by adding cold methanol.…”

Section: Methodsmentioning

confidence: 99%

Mild Hypoxia Promotes Survival and Proliferation of SOD2-Deficient Astrocytes via c-Myc Activation

et al. 2006

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Mouse astrocytes deficient in the mitochondrial form of manganese superoxide dismutase (SOD2) do not survive in culture under atmospheric air with 20% oxygen (O 2 ), which is a common condition for cell cultures. Seeding the cells and maintaining them under mild hypoxic conditions (5% O 2 ) circumvents this problem and allows the cells to grow and become confluent. Previous studies from our laboratory showed that this adaptation of the cells was not attributable to compensation by other enzymes of the antioxidant defense system. We hypothesized that transcriptional activity and upregulation of genes other than those with an antioxidant function are involved. Our present study shows that c-Myc was significantly induced and that it inhibited p21 and induced proteins such as cyclindependent kinases, cyclin D, and cyclin E, which are involved in the cell cycle process, along with phosphorylation of the retinoblastoma protein and Cdc2 (cell division cycle 2). These mechanisms contribute to cell proliferation. Small interfering RNA of c-Myc, however, blocked proliferation of SOD2 homozygous (SOD2Ϫ/Ϫ) astrocytes under mild hypoxia consisting of 5% O 2 , whereas it did not affect the growth of wild-type astrocytes. Our results indicate that c-Myc plays a critical role in hypoxia-induced proliferation and survival of SOD2Ϫ/Ϫ astrocytes by overcoming injury caused by oxidative stress.

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Section: Methodsmentioning

confidence: 99%

Mild Hypoxia Promotes Survival and Proliferation of SOD2-Deficient Astrocytes via c-Myc Activation

et al. 2006

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“…Neuronal cell viability was quantified by measuring MAP2 immunoreactivity. This method is favorable for measuring neuronal survival in the presence of glial cells without resorting to the counting of neurons (Brooke et al, 1999). Measurement was performed using monoclonal anti-MAP2 antibody and VECTASTAIN ABC-PO mouse IgG kit in accordance with the manufacturer's manual.…”

Section: Neuroprotective Effect Against A␤(1-42)-induced Toxicitymentioning

confidence: 99%

A Novel Neurotrophic Agent, T-817MA [1-{3-[2-(1-Benzothiophen-5-yl) Ethoxy] Propyl}-3-azetidinol Maleate], Attenuates Amyloid-β-Induced Neurotoxicity and Promotes Neurite Outgrowth in Rat Cultured Central Nervous System Neurons

Hirata

Yamaguchi²,

Takamura³

et al. 2005

J Pharmacol Exp Ther

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Progressive neuronal loss in Alzheimer's disease (AD) is considered to be a consequence of the neurotoxic properties of amyloid-␤ peptides (A␤). T-817MA (1-{3-[2-(1-benzothiophen-5-yl) ethoxy] propyl}-3-azetidinol maleate) was screened as a candidate therapeutic agent for the treatment of AD based on its neuroprotective potency against A␤-induced neurotoxicity and its effect of enhancing axonal regeneration in the sciatic nerve axotomy model. The neuroprotective effect of T-817MA against A␤(1-42) or oxidative stress-induced neurotoxicity was assessed using a coculture of rat cortical neurons with glia.

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“…The media in the inserts were supplemented with glucose (15 mM final glucose concentration), and the laminar cultures were incubated for 1 hr at 37°C to allow equilibration between the media in the inserts and that in the wells. Glutamate (5-20 M; predetermined weekly to give ϳ50% killing of neurons in the absence of vector) was spiked into the media, and 7 hr postinsult neuronal survival was determined using the 2,3Ј-azinobis(ethylbenzothiazoline-6-sulfonic) acid (ABTS) assay (Brooke et al, 1999). In brief, cells were immunostained with a MAP2 (neuronal marker) antibody (Sigma), a secondary antibody, followed by Vectastain ABC and ABTS kits (all from Vector Laboratories).…”

Section: Neurological Insults and Quantification Of Cell Survivalmentioning

confidence: 99%

Dual-Gene, Dual-Cell Type Therapy against an Excitotoxic Insult by Bolstering Neuroenergetics

Bliss¹,

Ip²,

Cheng³

et al. 2004

J. Neurosci.

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Increasing evidence suggests that glutamate activates the generation of lactate from glucose in astrocytes; this lactate is shuttled to neurons that use it as a preferential energy source. We explore this multicellular "lactate shuttle" with a novel dual-cell, dual-gene therapy approach and determine the neuroprotective potential of enhancing this shuttle. Viral vector-driven overexpression of a glucose transporter in glia enhanced glucose uptake, lactate efflux, and the glial capacity to protect neurons from excitotoxicity. In parallel, overexpression of a lactate transporter in neurons enhanced lactate uptake and neuronal resistance to excitotoxicity. Finally, overexpression of both transgenes in the respective cell types provided more protection than either therapy alone, demonstrating that a dual-cell, dual-gene therapy approach gives greater neuroprotection than the conventional single-cell, single-gene strategy.

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Quantification of neuron survival in monolayer cultures using an enzyme-linked immunosorbent assay approach, rather than by cell counting

Cited by 46 publications

References 15 publications

Mild Hypoxia Promotes Survival and Proliferation of SOD2-Deficient Astrocytes via c-Myc Activation

Mild Hypoxia Promotes Survival and Proliferation of SOD2-Deficient Astrocytes via c-Myc Activation

A Novel Neurotrophic Agent, T-817MA [1-{3-[2-(1-Benzothiophen-5-yl) Ethoxy] Propyl}-3-azetidinol Maleate], Attenuates Amyloid-β-Induced Neurotoxicity and Promotes Neurite Outgrowth in Rat Cultured Central Nervous System Neurons

Dual-Gene, Dual-Cell Type Therapy against an Excitotoxic Insult by Bolstering Neuroenergetics

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