Sensitive and reliable JC‐1 and TOTO‐3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: Interest for cell death investigations

Zuliani, Thomas; Duval, Raphaël E.; Jayat, Chantal; Schnébert, Sylviane; André, Patrice; Dumas, Marc; Ratinaud, Marie‐Hélène

doi:10.1002/cyto.a.10059

Cited by 66 publications

(54 citation statements)

References 43 publications

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“…Methods have been developed to estimate mitochondrial transmembrane potential modifications by FCM with cationic lipophilic dyes (28,29). Recently, JC-1 dye has been proposed to more accurately evaluate changes in mitochondrial activity (20,30). JC-1 is selectively incorporated into mitochondria in a monomeric form emitting at 527 nm after excitation at 488 nm with an argon laser.…”

Section: Discussionmentioning

confidence: 99%

“…TOTO-3 + ). Our FCM analyses were calibrated with 1 mM H 2 O 2 according to Zuliani et al (20). TOTO-3 staining was done in order to perform JC-1 staining on only those cells having intact cytoplasmic membranes (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…3). Moreover, JC-1/TOTO-3 double staining was recently described as providing a more reliable simultaneous assessment of the plasma membrane integrity and mitochondrial transmembrane potential occurring during apoptosis (20). As mitochondrial internal membrane potential dissipation is well-defined as an early apoptotic event (31), we decided to study this process after 24 h of UA treatment.…”

Section: Discussionmentioning

confidence: 99%

“…JC-1 indicates mitochondrial polarization whereas TOTO-3, a membrane impermeant probe, enters cells with altered plasma membrane integrity, binds to nucleic acids and exhibits strong red fluorescence, enabling discrimination between intact and altered cells. After UA treatment or, in the case of the controls, medium alone, HaCaT and M4Beu cells were stained according to Zuliani et al (20). Briefly, suspensions were adjusted to 10 6 cells/ml, stained for 35 min with 1 μg/ml JC-1 at 37˚C and then for 5 min with 1 μM TOTO-3 at 37˚C.…”

Section: Jc-1 and Toto-3 Staining For Flow Cytometric Analysismentioning

confidence: 99%

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Differential involvement of mitochondria during ursolic acid-induced apoptotic process in HaCaT and M4Beu cells

Duval¹,

Harmand²,

Jayat-Vignoles³

et al. 2008

Oncol Rep

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Abstract. Ursolic acid (UA) is a pentacyclic triterpenoid compound which exists widely in nature and is known to have a pleitropic biological activity profile. For the last few decades, extensive work has been carried out to establish its biological activities and pharmacological actions. It is described as a promising chemopreventive agent with an antiproliferative effect on cancer cells that stems from its ability to induce apoptosis. We investigated and compared the role played by mitochondria during the apoptotic process induced by UA in human HaCaT-derived keratinotic cells and M4Beu human melanoma cells. In both cell lines, UA induced significant caspase-3 activation, the downstream central effector of apoptosis. Subsequent JC-1/TOTO-3 double staining clearly demonstrated that UA induces strong mitochondrial-transmembrane potential collapse in M4Beu cells, while mitochondria from HaCaT-treated cells remain largely unstimulated. This was confirmed by Western blot analysis, which revealed a Bax/Bcl-2-balance change in favor of Bax, the proapoptotic member, in UA-treated M4Beu cells. It can be concluded that UA induces apoptosis in M4Beu through the mitochondrial pathway, while other mechanisms are activated in the case of HaCaT cells. IntroductionAmong natural compounds, ursolic acid (UA), the most frequently studied natural triterpenic acid (1), seems to be a promising chemical entity for the protection of human skin. Indeed, in cosmetology UA is often used for photoaging protection (2). It prevents and improves the appearance of wrinkles and age spots by restoring skin collagen structure and elasticity, stimulates collagen production in cultured fibroblasts and increases the production of ceramides in human epidermal keratinocytes and skin (3). UA may also be an effective inhibitor of the UVA-modulated signal transduction pathway in human HaCaT cells, in which it significantly suppresses UVA-induced reactive oxygen species production and lipid peroxidation (4). Moreover, it possesses anti-tumor properties including the inhibition of skin tumorigenesis (5) and tumor promotion (6). UA also induces apoptosis in several cancer cell lines (7-13).Previously, we studied the effect of UA on human HaCaTderived keratinotic cells and M4Beu human melanoma cells with the intention of confirming its role as a promising candidate for skin cancer prevention. In those studies, we demonstrated for the first time that UA has a significant antiproliferative effect associated with the induction of the apoptotic process (14,15). In the literature, it is well established that the activation of the apoptotic transduction pathway is dependent on cell type and subcellular targets. The two main apoptotic pathways are the extrinsic and intrinsic pathways (16). The extrinsic pathway is characterized by death ligand attachment on extracellular receptors (e.g. TNF/TNF-R) with a subsequent caspase-8 activation, which in turn cleaves and activates caspase-3 (17). The intrinsic pathway is triggered by different mitochondrial stresses and i...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Jc-1 and Toto-3 Staining For Flow Cytometric Analysismentioning

confidence: 99%

See 2 more Smart Citations

Differential involvement of mitochondria during ursolic acid-induced apoptotic process in HaCaT and M4Beu cells

Duval¹,

Harmand²,

Jayat-Vignoles³

et al. 2008

Oncol Rep

Self Cite

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“…JC-1 is a cationic dye that exhibits membrane potential-dependent accumulation in mitochondria, as indicated by a fluorescence emission shift from green (k 525 nm) to red (k 610 nm). Mitochondria depolarization is specifically indicated by a decrease in the red to green fluorescence intensity ratio [34]. Following OTA treatment cells were incubated with JC-1 following the manufacture's protocol of the JC-1 staining kit (Sigma).…”

Section: Mitochondrial Membrane Potential Assessment By Jc-1 Stainingmentioning

confidence: 99%

Ochratoxin A induces apoptosis in neuronal cells

et al. 2009

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The mycotoxin ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is a frequently present contaminant of food and feedstuffs. OTA exhibits a wide range of toxic activities including nephro-and hepatotoxicity. However, little is known regarding potential neurotoxic effects of OTA. In the present study primary neurons as well as SH-SY5Y neuronal cells were incubated with increasing concentrations of OTA (0.1-2.5 lmol/L). OTA treatment resulted in a dose-dependent increase in cytotoxicity in both neuronal cell types. Caspase-9 and caspase-3 were activated in response to OTA treatment. Furthermore, caspase inhibitors were effective in partly counteracting OTA induced neurocytotoxicity. OTA induced apoptosis was accompanied by a loss of mitochondria membrane potential. Overall, present data indicated that OTA is neurotoxic at relatively low concentrations. OTA induced neurotoxicity seems to be, at least party, mediated by apoptosis. OTA may contribute to the pathogenesis of neurodegenerative diseases (e.g. Alzheimer's and Parkinson's disease) in which apoptotic processes are centrally involved.

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Chemical Constituents of the Fruits of Cornus Officinalis and Evaluation of their Neuroprotective Activity

Yang,

Hao,

Wang

et al. 2024

Chemistry & Biodiversity

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The phytochemical investigation of the fruits of Cornus officinalis yielded a new phenolic acid derivative, neophenolic acid A (1), and a novel flavonoid glycoside, (2R)‐naringenin‐7‐O‐β‐(6″‐galloyl‐glucopyranoside) (2a), along with six known flavonoid glycosides (2b – 7). Their structures were determined by 1D, 2D NMR and HRESIMS data. The absolute configuration of 1 was established by ECD analysis. Compounds 1 – 7 were evaluated for their neuroprotective activities against corticosterone (CORT)‐induced injury in PC‐12 cells. Compounds 1, 2a, 2b, 5, and 6 exhibited neuroprotective activities against CORT‐induced neurotoxicity in PC‐12 cells. The underlying mechanism study suggested that compounds 1, 2a, 2b, 5, and 6 were able to attenuate CORT‐induced apoptosis and damage, increase the levels of MMP and decrease Ca2+ inward flow in PC‐12 cells.

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Sensitive and reliable JC‐1 and TOTO‐3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: Interest for cell death investigations

Cited by 66 publications

References 43 publications

Differential involvement of mitochondria during ursolic acid-induced apoptotic process in HaCaT and M4Beu cells

Differential involvement of mitochondria during ursolic acid-induced apoptotic process in HaCaT and M4Beu cells

Ochratoxin A induces apoptosis in neuronal cells

Chemical Constituents of the Fruits of Cornus Officinalis and Evaluation of their Neuroprotective Activity

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