Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry

Sureda, Francesc X.; Escubedo, Elena; Gabriel, C.; Comas, Jaume; Camarasa, Jorge; Camins, Antoni

doi:10.1002/(sici)1097-0320(19970501)28:1<74::aid-cyto9>3.0.co;2-h

Cited by 72 publications

(49 citation statements)

References 14 publications

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“…The mitochondrial membrane potential was assessed by the retention of rhodamine 123, a specific fluorescent cationic dye (Sureda 1997). Briefly, one milliliter of the cell suspension (10 6 cells) was loaded for 1 h with different concentrations of KA in a shaking bath at 37°C in an atmosphere of 95% O 2 /5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

U-83836E prevents kainic acid-induced neuronal damage

Camins

Gabriel

Aguirre

et al. 1998

Naunyn-Schmiedeberg's Arch Pharmacol

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The effect of kainic acid (KA) on mitochondrial membrane potential (MMP) and reactive-oxygen species (ROS) production was studied in dissociated cerebellar granule cells from rat pups. KA induced a maximum increase of 361%+/-35% in ROS production. The lazaroid compound U-83836E (at concentrations ranging from 10(-9) to 5x10(-6) M) completely inhibited this increase, with an IC50 value of 3.02+/-1.08x10(-7) M. KA also decreased the mitochondrial membrane potential (MMP), with a maximum decrease of about 30%. Absence of Na+ in the incubation medium did not significantly alter the effect of KA on MMP. As expected, the AMPA/kainate receptor antagonist NBQX inhibited the effects of KA on MMP with an IC50 value of 1.1+/-0.8 microM. However, the lazaroid U-83836E, indomethacin, nor-dihydroguaiaretic acid and L-nitroarginine all failed to inhibit the KA-induced decrease in the MMP. Finally, to assess the neuroprotective effect of U-83836E on KA-induced neurotoxicity in vivo, the increase in the peripheral-type benzodiazepine receptor density in rat hippocampus was measured. Treatment with KA increased the Bmax to 1341+/-192 fmol mg(-1). When U-83836E was coadministered with KA, the Bmax was reduced to 765+/-122 fmol mg(-1), which was not significantly different from the Bmax obtained from untreated rats (Bmax: 518+/-33 fmol mg(-1)). We conclude that treatment with the lazaroid U-83836E might be a suitable therapeutic strategy in neurodegenerative disorders.

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Section: Methodsmentioning

confidence: 99%

U-83836E prevents kainic acid-induced neuronal damage

Camins

Gabriel

Aguirre

et al. 1998

Naunyn-Schmiedeberg's Arch Pharmacol

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“…Rhodamine 123 is a cationic lipophilic fluorochrome whose distribution to the mitochondria matrix correlates with the DW m (15). After treatment, cells were loaded with 10 Amol/L rhodamine 123 and incubated at 37jC for 30 minutes in the dark.…”

Section: Measurement Of Mitochondrial Membrane Potentialmentioning

confidence: 99%

Pristimerin induces caspase-dependent apoptosis in MDA-MB-231 cells via direct effects on mitochondria

Chan

Chen

et al. 2005

Molecular Cancer Therapeutics

115

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“…The latter are mostly based on the Dw-dependent accumulation of fluorescent cations within the mitochondrial matrix. Of these cations, rhodamine 123 (R123), the methylester (TMRM) and ethylester (TMRE) of tetramethylrhodamine, and the ratiometric indicator 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) have been widely applied (25)(26)(27)(28). The fluorescence of these compounds can be read out using fluorimetry of cell suspensions in a cuvette, flow cytometry of individual cells, or fluorescence microscopy to obtain subcellular information.…”

Section: Discussionmentioning

confidence: 99%

Life cell quantification of mitochondrial membrane potential at the single organelle level

et al. 2007

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Mitochondrial membrane potential (Dw) is key to mitochondrial function and cellular survival. Here, we aimed to develop an automated protocol allowing sensitive quantification of Dw in living cells at the level of individual mitochondria. Human skin fibroblasts were stained with the fluorescent cation tetramethyl rhodamine methyl ester (TMRM), which is sequestered by mitochondria according to their Dw. Cells were visualized by videomicroscopy and the acquired images were processed to generate a mitochondria-specific mask. The latter was superimposed on the original image to allow quantification of TMRM fluorescence. Following validation, our approach revealed that mitochondria with different Dw coexisted within the same cell. Furthermore, our method allowed reproducible detection of small (\10%) reductions in TMRM intensity induced by the complex III inhibitor antimycin A. Mitochondrial uncoupling by p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) greatly reduced mitochondrial TMRM fluorescence. Under these conditions faithful mask calculation and TMRM intensity analysis were still possible using a mitochondria-targeted green fluorescence protein (mitoAcGFP1), expressed in the cells using baculoviral transfection. ' International Society for Analytical CytologyKey terms fibroblasts; TMRM; AcGFP1; image processing and analysis MITOCHONDRIA actively maintain a highly negative potential across their inner membrane (Dw), which is essential for mitochondrial function and cell viability. This potential is maintained by four protein complexes (CI-CIV) of the mitochondrial electron transport chain (ETC) that, together with the F 0 /F 1 -ATP-synthase (CV), constitute the oxidative phosphorylation (OXPHOS) system (1). Mitochondria are fuelled by pyruvate and fatty acids, which are used as carbon sources for the tricarboxylic acid cycle in the mitochondrial matrix. The products of this cycle, NADH and FADH 2 , subsequently feed electrons into the ETC at CI and CII, respectively. At three locations within the chain, CI, CIII, and CIV, the electron transport is used to expel protons from the matrix space to the intermembrane space. The H 1 ejection results in the establishment of an electrochemical H 1 gradient, which consists of a chemical (DpH) and electrical (Dw) component. At CV, protons are allowed to flow back into the mitochondrial matrix to drive the synthesis of ATP from ADP and inorganic phosphate (P i ). In intact respiring mitochondria, the ETC and ATP synthesis are efficiently coupled. Importantly, a large number of diseases have been linked to mitochondrial dysfunction, including age-related neurological diseases, cardiovascular disease, cancer and diabetes (reviewed in Refs. 2, 3).Since Dw is regarded as a key indicator of mitochondrial health and metabolic activity, the accurate determination of this parameter in intact cells is important to address many research questions. Most assays for monitoring Dw in living cells

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Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry

Cited by 72 publications

References 14 publications

U-83836E prevents kainic acid-induced neuronal damage

U-83836E prevents kainic acid-induced neuronal damage

Pristimerin induces caspase-dependent apoptosis in MDA-MB-231 cells via direct effects on mitochondria

Life cell quantification of mitochondrial membrane potential at the single organelle level

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