scholarly journals Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

10.3791/55166 ◽  
2017 ◽  
Author(s):  
Matthew McKenzie ◽  
Sze C. Lim ◽  
Michael R. Duchen
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Simultaneous Measurement ◽  
Mitochondrial Membrane ◽  
Fluorescent Microscopy ◽  
Live Cells ◽  
Mitochondrial Calcium

scholarly journals Mechanism of apoptosis induced by quinoxalone from the myxobacterium Stigmatella eracta WXNXJ-B in B16 mouse melanoma cell line

2017 ◽  
Author(s):  
Dahong Wang ◽  
Lanlan Wei ◽  
Shuaiying Zhang
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Biological Activities ◽  
Chromatin Condensation ◽  
Fluorescent Microscopy ◽  
Dependent Manner ◽  
Dna Ladder ◽  
Mouse Melanoma ◽  
B16 Mouse Melanoma

The biological activities of quinoxalone, a novel small molecular substance isolated from the broth of the myxobacterium Stigmatella eracta WXNXJ-B, was investigated. This study was designed to determine the anti-proliferative, apoptotic property of quinoxalone, using B16 mouse melanoma cells as a model system. The results showed that quinoxalone has antitumor activity and can significantly inhibit the proliferation of B16 cells. The extent and the timing of apoptosis were strongly dependent on the dose. After treating with quinoxalone and staining with Hoechst 33342, B16 cells showed typical apoptotic morphological features such as chromatin condensation by fluorescent microscopy. DNA isolated from B16 cells cultured with quinoxalone showed a typical DNA ladder of apoptosis in agarose gel electrophoresis. Further investigation results showed that the apoptotic machinery of B16 induced by quinoxalone was associated with drop in mitochondrial membrane potential from 5.35% to 23.7%, up-regulation of Bax and down-regulation of Bcl-2 in a dose-dependent manner. And a significant increased activation of caspase-3. Our finding suggests that quinoxalone could suppress the growth of B16 cells and reduces cell survival via disturbing mitochondrial membrane potential and inducing apoptosis of tumor cells.

scholarly journals Expression of the mitochondrial calcium uniporter in cardiac myocytes improves impaired mitochondrial calcium handling and metabolism in simulated hyperglycemia

2016 ◽  
Vol 311 (6) ◽  
pp. C1005-C1013 ◽  
Author(s):  
Julieta Diaz-Juarez ◽  
Jorge Suarez ◽  
Federico Cividini ◽  
Brian T. Scott ◽  
Tanja Diemer ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Cardiac Myocytes ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Calcium Handling ◽  
Acid Oxidation ◽  
Mitochondrial Calcium ◽  
Pathophysiological Mechanism ◽  
Protein Levels

Diabetic cardiomyopathy is associated with metabolic changes, including decreased glucose oxidation (Gox) and increased fatty acid oxidation (FAox), which result in cardiac energetic deficiency. Diabetic hyperglycemia is a pathophysiological mechanism that triggers multiple maladaptive phenomena. The mitochondrial Ca2+ uniporter (MCU) is the channel responsible for Ca2+ uptake in mitochondria, and free mitochondrial Ca2+ concentration ([Ca2+]m) regulates mitochondrial metabolism. Experiments with cardiac myocytes (CM) exposed to simulated hyperglycemia revealed reduced [Ca2+]m and MCU protein levels. Therefore, we investigated whether returning [Ca2+]m to normal levels in CM by MCU expression could lead to normalization of Gox and FAox with no detrimental effects. Mouse neonatal CM were exposed for 72 h to normal glucose [5.5 mM glucose + 19.5 mM mannitol (NG)], high glucose [25 mM glucose (HG)], or HG + adenoviral MCU expression. Gox and FAox, [Ca2+]m, MCU levels, pyruvate dehydrogenase (PDH) activity, oxidative stress, mitochondrial membrane potential, and apoptosis were assessed. [Ca2+]m and MCU protein levels were reduced after 72 h of HG. Gox was decreased and FAox was increased in HG, PDH activity was decreased, phosphorylated PDH levels were increased, and mitochondrial membrane potential was reduced. MCU expression returned these parameters toward NG levels. Moreover, increased oxidative stress and apoptosis were reduced in HG by MCU expression. We also observed reduced MCU protein levels and [Ca2+]m in hearts from type 1 diabetic mice. Thus we conclude that HG-induced metabolic alterations can be reversed by restoration of MCU levels, resulting in return of [Ca2+]m to normal levels.

scholarly journals Mechanism of apoptosis induced by quinoxalone from the myxobacterium Stigmatella eracta WXNXJ-B in B16 mouse melanoma cell line

2017 ◽  
Author(s):  
Dahong Wang ◽  
Lanlan Wei ◽  
Shuaiying Zhang
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Biological Activities ◽  
Chromatin Condensation ◽  
Fluorescent Microscopy ◽  
Dependent Manner ◽  
Dna Ladder ◽  
Mouse Melanoma ◽  
B16 Mouse Melanoma

The biological activities of quinoxalone, a novel small molecular substance isolated from the broth of the myxobacterium Stigmatella eracta WXNXJ-B, was investigated. This study was designed to determine the anti-proliferative, apoptotic property of quinoxalone, using B16 mouse melanoma cells as a model system. The results showed that quinoxalone has antitumor activity and can significantly inhibit the proliferation of B16 cells. The extent and the timing of apoptosis were strongly dependent on the dose. After treating with quinoxalone and staining with Hoechst 33342, B16 cells showed typical apoptotic morphological features such as chromatin condensation by fluorescent microscopy. DNA isolated from B16 cells cultured with quinoxalone showed a typical DNA ladder of apoptosis in agarose gel electrophoresis. Further investigation results showed that the apoptotic machinery of B16 induced by quinoxalone was associated with drop in mitochondrial membrane potential from 5.35% to 23.7%, up-regulation of Bax and down-regulation of Bcl-2 in a dose-dependent manner. And a significant increased activation of caspase-3. Our finding suggests that quinoxalone could suppress the growth of B16 cells and reduces cell survival via disturbing mitochondrial membrane potential and inducing apoptosis of tumor cells.

scholarly journals Mitochondrial Dysfunction in Chronic Respiratory Diseases: Implications for the Pathogenesis and Potential Therapeutics

2021 ◽  
Vol 2021 ◽  
pp. 1-20
Author(s):  
Wen-cheng Zhou ◽  
Jiao Qu ◽  
Sheng-yang Xie ◽  
Yang Sun ◽  
Hong-wei Yao
Keyword(s):  
Mitochondrial Dna ◽  
Membrane Potential ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Respiratory Diseases ◽  
Metabolic Reprogramming ◽  
Mitochondrial Calcium ◽  
Chronic Respiratory Diseases ◽  
Cellular Processes

Mitochondria are indispensable for energy metabolism and cell signaling. Mitochondrial homeostasis is sustained with stabilization of mitochondrial membrane potential, balance of mitochondrial calcium, integrity of mitochondrial DNA, and timely clearance of damaged mitochondria via mitophagy. Mitochondrial dysfunction is featured by increased generation of mitochondrial reactive oxygen species, reduced mitochondrial membrane potential, mitochondrial calcium imbalance, mitochondrial DNA damage, and abnormal mitophagy. Accumulating evidence indicates that mitochondrial dysregulation causes oxidative stress, inflammasome activation, apoptosis, senescence, and metabolic reprogramming. All these cellular processes participate in the pathogenesis and progression of chronic respiratory diseases, including chronic obstructive pulmonary disease, pulmonary fibrosis, and asthma. In this review, we provide a comprehensive and updated overview of the impact of mitochondrial dysfunction on cellular processes involved in the development of these respiratory diseases. This not only implicates mechanisms of mitochondrial dysfunction for the pathogenesis of chronic lung diseases but also provides potential therapeutic approaches for these diseases by targeting dysfunctional mitochondria.

23 REDUCTION OF MITOCHONDRIAL FUNCTION, PROLIFERATION, AND GENE EXPRESSION IN FIBROBLAST DONOR CELLS FOR USE IN SOMATIC CELL NUCLEAR TRANSFER BY CPI-613 AND PS48

2016 ◽  
Vol 28 (2) ◽  
pp. 141
Author(s):  
B. R. Mordhorst ◽  
S. L. Murphy ◽  
L. D. Spate ◽  
R. M. Ross ◽  
K. D. Wells ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Somatic Cell ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Tricarboxylic Acid Cycle ◽  
Pi3k Pathway ◽  
Live Cells ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Main Effect

The morphology (spherical and without cristae) and metabolism (lowly functional) of mitochondria in early embryos and other rapidly proliferating cells exhibit a Warburg effect (WE)-like metabolism. A hallmark of the WE is the predominate use of glycolysis for energy production as opposed to the tricarboxylic acid cycle used by differentiated cells. Additionally, increased signalling of the PI3K pathway is correlated with an increase in glucose metabolism within cancer cells and is consistent with the WE. PS48 stimulates the PI3K pathway, and CPI-613 inhibits pyruvate dehydrogenase. The goal was to achieve a WE-like effect in donor cells before NT. Day 35 porcine fetal fibroblasts were treated as controls (CON, 0 μM) or with CPI (25, 50, or 100 μM) or PS48 (1, 5, 10 μM) for 7 days. Cytometry data were processed using SUMMIT software and analysed via GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA); all variables were analysed for the main effect of drug concentration. Trypan blue cell viability measures were analysed using GLM. For each collection day (i.e. Day 3, 5, and 7), all variables were analysed for the main effect of treatment, duration of culture, and their interaction. All mRNA expression as measured via the ΔΔ-ct method by qPCR was analysed using CT values in GLM for the main effect of drug treatment. Total number of cells and live cells at 120 h was decreased (P ≤ 0.03) in all PS48 treatments compared with CON cells (total cells: CON = 8.95 × 106 v. PS48 treatments ≤6.98 × 106; live cells: CON = 8.39 × 106 v. PS48 treatments ≤6.50 × 106). While the percentage live cells in CPI and CON cells did not differ (P ≥ 0.09), 100 μM decreased the number of total cells and live cells from that of CON for every time point by ~50% (P ≤ 0.02), whereas the other CPI treatments 25 and 50 μM were intermediate. Expression of PDK2 was reduced with 10 μM PS48 treatment compared with CON, and 50 and 100 μM CPI treated cells (P < 0.001; PS48 10 μM: 0.335 v. ≤1.012 other treatments). The CPI 100 μM and 10 μM PS48 concentrations decreased PKM M1 variant expression compared with CON and 50 μM CPI cells (P < 0.001; CPI 100 μM and PS48 10 μM <0.44; CPI 50 μM and CON >0.68). To determine the mitochondrial membrane potential, JC-10 was used. The percentage of cells with high mitochondrial membrane potential decreased (P = 0.04) with PS48 treatment (PS48 treatments ≤19.6%, control = 25.6%). Treatment with CPI also decreased (P ≤ 0.01) membrane potential and the percentages of cells (high function: CPI treatments ≤12.7 v. 25.6% in control; low function: CPI treatments ≥80.3 v. 74.3%). Because PS48 or CPI decrease mitochondrial membrane potential and the abundance of PKM M1, the metabolism of these potential donor cells may be more blastomere like. Experiments are underway to determine whether cells treated with PS48 or CPI will result in better development after somatic cell NT. This study was funded by Food for the 21st Century and NIH R01HD080636.

scholarly journals Phenotypic Changes in Mitochondrial Membrane Potential (Δψm) during Valinomycin-Induced Depolarisation and Apoptosis

2005 ◽  
Vol 27 (4) ◽  
pp. 231-236
Author(s):  
M. L. Morrison ◽  
K. Williamson ◽  
K. Arthur ◽  
G. J. Price ◽  
P. W. Hamilton ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Texture Analysis ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Fluorescent Microscopy ◽  
Large Body ◽  
Texture Features ◽  
Sw620 Cell ◽  
Phenotypic Changes ◽  
Induced Apoptosis

A large body of evidence has implicated mitochondria in control of cell death, where key apoptotic mechanisms involve change in mitochondrial membrane permeability and depolarisation of mitochondrial membrane potential (Δψm). Assessment of Δψm is traditionally conducted using the lipophilic cation JC-1 on the flow cytometer or by fluorescent microscopy. Here we assess JC-1 aggregation using the novel tool of digital texture analysis to establish mitochondrial phenotypic changes induced by the K+ ionophore, valinomycin in a unique model comprising SW480 and SW620 cell lines. This provides an opportunity to study these phenomena in the context of colorectal cancer. Valinomycin-induced apoptosis was detected using morphology and analysis of DNA content. Cells were treated with valinomycin, images digitally recorded on a calibrated video photometer and subjected to high resolution digital texture analysis. This demonstrated that the HARAM texture features (Mean of the Haralick texture features) were highly valuable in describing the transition of Δψm as the cell undergoes apoptosis. In Conclusion this study illustrates the potential of texture analysis as a novel and additional technique for quantifying JC-1 aggregation and revealing the spectrum of collapse of Δψm during apoptosis.

scholarly journals Is the Mitochondrial Membrane Potential (∆Ψ) Correctly Assessed? Intracellular and Intramitochondrial Modifications of the ∆Ψ Probe, Rhodamine 123

2022 ◽  
Vol 23 (1) ◽  
pp. 482
Author(s):  
Ljubava D. Zorova ◽  
Evgeniya A. Demchenko ◽  
Galina A. Korshunova ◽  
Vadim N. Tashlitsky ◽  
Savva D. Zorov ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Fluorescent Microscopy ◽  
Rhodamine 123 ◽  
Proton Pumps ◽  
Hydrogen Ions ◽  
Mandatory Requirement ◽  
Electrical Component

The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe.

scholarly journals Disruption of Neurotransmission, Membrane Potential, and Mitochondrial Calcium in the Brain and Spinal Cord of Nile Tilapia Elicited by Microcystis Aeruginosa Extract: An Uncommon Consequence of the Eutrophication Process

2021 ◽  
Author(s):  
Minerva Nájera-Martínez ◽  
Goretti Guadalupe Landon-Hernández ◽  
José Pablo Romero-López ◽  
María Lilia Domínguez-López ◽  
Ethel Awilda García-Latorre ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Protein Phosphatase ◽  
Nile Tilapia ◽  
Mitochondrial Membrane ◽  
Positive Control ◽  
Mitochondrial Calcium ◽  
Brain And Spinal Cord ◽  
The Brain

Abstract Microcystins (MCs) are produced during the growth and proliferation of some species of cyanobacteria, mainly Microcystis aeruginosa, which has massive growth in eutrophic water bodies. Microcystins are highly toxic Microcystis-derived metabolites that exert its main effect in the liver through the inhibition of protein phosphatase (PP1 and PP2A). However, other damages in fish species are less documented and could be unexpected. The aim of the current study was to evaluate the effects of Microcystis aeruginosa extract (MaE) into the central nervous system (CNS) of the Nile tilapia. The MaE was normalized by MCs content (MC-LR). We include a positive control for protein phosphatase inhibition, norcantharidin intraperitoneally dosed at sublethal levels. On the eighth day, measurement of neurotransmission biomarkers (AChE, BChE, CbE and GABA) were measured, as well as levels of mitochondrial calcium, and the mitochondrial membrane potential by flow cytometry in the brain and spinal cord were assessed, in addition to the PP1/PP2A activity in the liver. The MCs elicited mortality at 5 µg/L. The positive control and MCs at sublethal levels inhibited the PP1/PP2A activity in the liver and induced alterations in the neurotoxicity biomarkers evaluated in the CNS. This response is probably due to the disruption of transport ions, dependent and independent of ATP because of alterations in the mitochondrial membrane potential and mitochondrial calcium. The findings of this study suggest that pollutants capable of inducing cyanobacterial blooms are able, in an indirect way, to exert neurotoxic effects in fish species through MC levels.

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