The MTT-formazan assay: Complementary technical approaches and in vivo validation in Drosophila larvae

Pascua-Maestro, Raquel; Corraliza-Gomez, Miriam; Díez-Hermano, Sergio; PEREZ-SEGURADO, CANDIDO; Ganfornina, Marı́a D.; Sánchez, Diego

doi:10.1016/j.acthis.2018.01.006

Cited by 39 publications

(22 citation statements)

References 14 publications

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“…The MTT assay is a colorimetric cytotoxicity assay and one of the most widely employed viability assays in biomedical research . This assay is a cost effective and rapid method for investigating cell viability or proliferation . The reduction reaction of the tetrazolium compound to formazan by cellular enzymes, including mitochondrial NAD(P)H‐dependent oxidoreductases and dehydrogenases, is lowered in a population of cells exposed to a toxic compound .…”

Section: Resultsmentioning

confidence: 99%

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells

et al. 2018

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Suppression of the expression or activities of enzymes that are involved in the synthesis of de novo lipogenesis ( DNL ) in cancer cells triggers cell death via apoptosis. The plasma membrane citrate transporter ( PMCT ) is the initial step that translocates citrate from blood circulation into the cytoplasm for de novo long‐chain fatty acids synthesis. This study investigated the antitumor effect of the PMCT inhibitor ( PMCT i) in inducing apoptosis by inhibiting the DNL pathway in HepG2 cells. The present findings showed that PMCT i reduced cell viability and enhanced apoptosis through decreased intracellular citrate levels, which consequently caused inhibition of fatty acid and triacylglycerol productions. Thus, as a result of the reduction in fatty acid synthesis, the activity of carnitine palmitoyl transferase‐1 ( CPT ‐1) was suppressed. Decreased CPT ‐1 activity also facilitated the disruption of mitochondrial membrane potential (ΔΨm) leading to stimulation of apoptosis. Surprisingly, primary human hepatocytes were not affected by PMCT i. Increased caspase‐8 activity as a consequence of reduction in fatty acid synthesis was also found to cause disruption of ΔΨm. In addition, apoptosis induction by PMCT i was associated with an enhanced reactive oxygen species generation. Taken together, we suggest that inhibition of the DNL pathway following reduction in citrate levels is an important regulator of apoptosis in HepG2 cells via suppression of CPT ‐1 activity. Thus, targeting the DNL pathway mediating CPT ‐1 activity by PMCT i may be a selective potential anticancer therapy.

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Section: Resultsmentioning

confidence: 99%

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells

et al. 2018

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“…Cell viability was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay as previously described (Bajo-Grañeras et al, 2011; Pascua-Maestro et al, 2018). After MTT exposure for 3 h, cells were incubated in isopropanol with 10% Triton X-100, and the solubilized formazan was measured by spectrophotometry using the SOFTmax Pro microplate reader (Molecular Devices).…”

Section: Methodsmentioning

confidence: 99%

Extracellular Vesicles Secreted by Astroglial Cells Transport Apolipoprotein D to Neurons and Mediate Neuronal Survival Upon Oxidative Stress

Pascua-Maestro

González

Lillo

et al. 2019

Front. Cell. Neurosci.

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124

122

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Extracellular vesicle (EV)-mediated glia-to-neuron communication has been recognized in a growing number of physiological and pathological situations. They transport complex sets of molecules that can be beneficial or detrimental for the receiving cell. As in other areas of biology, their analysis is revolutionizing the field of neuroscience, since fundamental signaling processes are being re-evaluated, and applications for neurodegenerative disease therapies have emerged. Using human astrocytic and differentiated neuronal cell lines, we demonstrate that a classical neuroprotective protein, Apolipoprotein D (ApoD), expressed by glial cells and known to promote functional integrity and survival of neurons, is exclusively transported by EVs from astrocytes to neurons, where it gets internalized. Indeed, we demonstrate that conditioned media derived from ApoD-knock-out (KO) astrocytes exert only a partial autocrine protection from oxidative stress (OS) challenges, and that EVs are required for ApoD-positive astrocytic cell line derived medium to exert full neuroprotection. When subfractionation of EVs is performed, ApoD is revealed as a very specific marker of the exosome-containing fractions. These discoveries help us reframe our understanding of the neuroprotective role of this lipid binding protein and open up new research avenues to explore the use of systemically administered ApoD-loaded exosomes that can cross the blood-brain barrier to treat neurodegenerative diseases.

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“…To measure cell viability, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was performed as previously described [ 39 ]. Briefly, cells were seeded in a 96-well standard plate (NUNC, Thermo Scientific), with a cell density of 450 cells/mm 2 , in quadruplicates.…”

Section: Methodsmentioning

confidence: 99%

Modulation of Glial Responses by Furanocembranolides: Leptolide Diminishes Microglial Inflammation in Vitro and Ameliorates Gliosis In Vivo in a Mouse Model of Obesity and Insulin Resistance

et al. 2020

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Neurodegenerative diseases are age-related disorders caused by progressive neuronal death in different regions of the nervous system. Neuroinflammation, modulated by glial cells, is a crucial event during the neurodegenerative process; consequently, there is an urgency to find new therapeutic products with anti-glioinflammatory properties. Five new furanocembranolides (1−5), along with leptolide, were isolated from two different extracts of Leptogorgia sp., and compound 6 was obtained from chemical transformation of leptolide. Their structures were determined based on spectroscopic evidence. These seven furanocembranolides were screened in vitro by measuring their ability to modulate interleukin-1β (IL-1β) production by microglial BV2 cells after LPS (lipopolysaccharide) stimulation. Leptolide and compounds 3, 4 and 6 exhibited clear anti-inflammatory effects on microglial cells, while compound 2 presented a pro-inflammatory outcome. The in vitro results prompted us to assess anti-glioinflammatory effects of leptolide in vivo in a high-fat diet-induced obese mouse model. Interestingly, leptolide treatment ameliorated both microgliosis and astrogliosis in this animal model. Taken together, our results reveal a promising direct biological effect of furanocembranolides on microglial cells as bioactive anti-inflammatory molecules. Among them, leptolide provides us a feasible therapeutic approach to treat neuroinflammation concomitant with metabolic impairment.

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The MTT-formazan assay: Complementary technical approaches and in vivo validation in Drosophila larvae

Cited by 39 publications

References 14 publications

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells

Extracellular Vesicles Secreted by Astroglial Cells Transport Apolipoprotein D to Neurons and Mediate Neuronal Survival Upon Oxidative Stress

Modulation of Glial Responses by Furanocembranolides: Leptolide Diminishes Microglial Inflammation in Vitro and Ameliorates Gliosis In Vivo in a Mouse Model of Obesity and Insulin Resistance

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