Quantification of Reactive Oxygen Species Using 2&amp;#8242;,7&amp;#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&amp;#252;ller Glial Cells

Vaglienti, María V.; Subirada, Paula V.; Barcelona, Pablo F.; Bonacci, Gustavo; Sanchez, M. C.

doi:10.3791/63337-v

Cited by 3 publications

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“…BoNT and OXA‐mediated Ca 2+ influx finally led to tumour cell apoptosis and death through the increase of mROS, ΔΨm and cROS in tumour cells 4,12 . The BoNT and OXA‐dependent increases in mROS, ΔΨm and cROS were evaluated in HT29 cells loaded with MitoSOX, JC‐1 and DCFH‐DA, the most widely used fluorescent dyes to monitor mROS, ΔΨm and cROS levels 31,32 . The mitochondria in the JC‐1 analyses were marked by using the Mito Tracker red dye.…”

Section: Resultsmentioning

confidence: 99%

“…4,12 The BoNT and OXA-dependent increases in mROS, ΔΨm and cROS were evaluated in HT29 cells loaded with MitoSOX, JC-1 and DCFH-DA, the most widely used fluorescent dyes to monitor mROS, ΔΨm and cROS levels. 31,32 The mitochondria in the JC-1 analyses were marked by using the Mito Tracker red dye. The CCD fluorescein camera was used for the analyses of Mito Tracker Red, JC-1 (orange), BF, overlay (Figure 5A) and 2.5D (Figure 5B), although CLSM microscope was used to acquire MitoSOX (red), DCFH-DA (green) and their overlay (Figure 6A), as well as 2.5D images (Figure 6B).…”

Section: Inhibiting Trpm2 Reduced the Rise In Ht29 Death Induced By B...mentioning

confidence: 99%

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Synergic actions of botulinum neurotoxin A and oxaliplatin on colorectal tumour cell death through the upregulation of TRPM2 channel‐mediated oxidative stress

Demir,

Duman,

Nazıroğlu

2024

Clin Exp Pharma Physio

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Botulinum neurotoxin A (BoNT) is being shown to have anticancer action as a potential adjuvant treatment. The transient receptor potential (TRP) melastatin 2 (TRPM2) stimulator action of BoNT was reported in glioblastoma cells, but not in colorectal cancer (HT29) cells. By activating TRPM2, we evaluated the impacts of BoNT and oxaliplatin (OXA) incubations on oxidant and apoptotic values within the HT29 cells. Control, BoNT (5 IU for 24 h), OXA (50 μM for 24 h) and their combinations were induced. We found that TRPM2 protein is upregulated and mediates enhanced BoNT and OXA‐induced Ca2+ entry in cells as compared to control cells. The increase of free reactive oxygen species (ROS), but the decrease of glutathione is the main ROS responsible for TRPM2 activation on H29 exposure to oxidative stress. BoNT and OXA‐mediated Ca2+ entry through TRPM2 stimulation in response to H2O2 results in mitochondrial Ca2+ overload, followed by mitochondrial membrane depolarization, apoptosis and caspase‐3/‐8/‐9, although they were diminished in the TRPM2 antagonist groups (N‐(p‐amylcinnamoyl)anthranilic acid and carvacrol). In conclusion, by increasing the susceptibility of HT29 tumour cells to oxidative stress and apoptosis, the combined administration of BoNT and OXA via the targeting of TRPM2 may offer a different approach to kill the tumour cells.

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Section: Resultsmentioning

confidence: 99%

Section: Inhibiting Trpm2 Reduced the Rise In Ht29 Death Induced By B...mentioning

confidence: 99%

Synergic actions of botulinum neurotoxin A and oxaliplatin on colorectal tumour cell death through the upregulation of TRPM2 channel‐mediated oxidative stress

Demir,

Duman,

Nazıroğlu

2024

Clin Exp Pharma Physio

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“…For twenty-five minutes at 37°C in the dark, the cells were stained with 2 mM non-fluorescence MitoSOX, JC1, and DCFH-DA. Non-fluorescent DCFH-DA undergoes oxidation in the cytosol to produce fluorescent 2',7'dichlorofluorescein (DCF)(Vaglienti et al 2022).MitoSOX, JC-1, and DCF pictures were taken with the LSCM-800. DCF, MitoSOX, and JC-1 have excitation and emission wavelengths of 504/525 nm, 576/598 nm, and 593/595 nm, respectively.…”

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confidence: 99%

Neuroprotective action of honey bee venom (melittin) against hypoxiainduced oxidative toxicity and cell death via inhibition of the TRPM2 channel

ERTİLAV

2023

Journal of Cellular Neuroscience and Oxidative Stress

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One bioactive element of honeybee venom is melittin (MEL). MEL induced oxidant and apoptotic activities through the increase of mitochondrial Zn2+ and Ca2+ in tumor cells, but it also induced neuroprotective activity by inhibiting the cell death, intracellular reactive oxygen species (iROS), and mitochondrial ROS (mROS) productions in neurons. By stimulating the TRPM2 channel, hypoxia (HPO) enhances the effects of oxidative stress and neuronal death; however, its inhibition prevents the alterations. I studied the neuroprotective effect of MEL on HPO-mediated oxidative neurotoxicity and cell death in SH-SY5Y neuronal cells by altering the TRPM2 signaling pathways. In the SH-SY5Y cells, five groups were induced as control, MEL (1 ug/ml for 24 hrs), HPO (CoCl2 and 200 M for 24 hrs), HPO + MEL, and HPO + TRPM2 antagonist (2-aminoethoxydiphenyl borate, 2APB) (100 M for 2 hrs). The amounts of cytosolic free Ca2+ were increased in the HPO group by the stimulation of hydrogen peroxide, although they were decreased in the cells by the treatment of 2APB and MEL. The amount of cytosolic free Ca2+ was higher in the HPO group than in the control group. The amounts of cell death (propidium iodide positive cell number), oxidants (mROS and iROS), mitochondrial membrane depolarization, and cytosolic free Zn2+ were higher in the HPO group than in the control and MEL groups, although their amounts were lower in the HPO + MEL and HPO + 2APB groups than in the HPO group only. In conclusion, MEL therapy reduced the amount of HPO-induced oxidative stress and neuronal deaths in SH-SY5Y cells by inhibiting TRPM2. The MEL could be considered as a potential protective component against oxidative neuronal damage caused by HPO.

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Propofol's Neuroprotective Effect Against Cisplatin-Induced Oxidative Neurotoxicity Via Suppression of the TRPM2 Cation Channel

Osmanlıoğlu

2024

Online Türk Sağlık Bilimleri Dergisi

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Objective: Cisplatin (CSP) exhibits strong oxidant and apoptotic effects in tumors, but it also causes adverse neurodegenerative effects by stimulating the TRPM2 cation channel. By regulating mitochondrial reactive free oxygen species (ROS) and excessive Ca2+ entry-mediated apoptosis, propofol (PRPF) exhibits antioxidant and neuroprotective properties. However, the action of the TRPM2 in these productions in human SH-SY5Y neuronal cells has not yet been determined. In SH-SY5Y, I investigated the protective effects of PRPF by modifying TRPM2, which affects CSP-induced neuronal mitochondrial function and death. Materials and Methods: I generated five main groups in the SH-SY5Y as control, PRPF (200 mM for 24h), CSP (25 mM for 24h), CSP + PRPF, and CSP + TRPM2 channel antagonists (25 mM ACA and 100 mM 2APB). Results: Through TRPM2 stimulation, the incubation with CSP increased the amounts of apoptosis, caspase -3, caspase -9, cell death percentage, ROS, mitochondrial hyperpolarization, TRPM2 current densities, and intracellular free Ca2+. However, the incubation of PRPF through the inhibition of TRPM2 decreased the amounts of these processes. Conclusions: PRPF treatment via TRPM2 suppression decreased the levels of mitochondrial oxidative stress and neuronal death caused by CSP. One effective therapy option for CSP-induced mitochondrial oxidative neuronal damage is the PRPF.

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Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells

Cited by 3 publications

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Synergic actions of botulinum neurotoxin A and oxaliplatin on colorectal tumour cell death through the upregulation of TRPM2 channel‐mediated oxidative stress

Synergic actions of botulinum neurotoxin A and oxaliplatin on colorectal tumour cell death through the upregulation of TRPM2 channel‐mediated oxidative stress

Neuroprotective action of honey bee venom (melittin) against hypoxiainduced oxidative toxicity and cell death via inhibition of the TRPM2 channel

Propofol's Neuroprotective Effect Against Cisplatin-Induced Oxidative Neurotoxicity Via Suppression of the TRPM2 Cation Channel

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