Changes in mitochondrial transmembrane potential ([DELTA][psi]m) were analyzed using a TMRE (tetramethylrhodamine, ethyl ester) assay .
We used a TMRE assay, which quantifies changes in mitochondrial membrane potential in live cells, and a cell-permeable, positively charged, red-orange dye that readily accumulates in active mitochondria due to their relative negative charge.
The mitochondrial membrane potential ([DELTA][PSI]m) of H9c2 cardiac cells was then assessed with tetramethylrhodamine ethyl ester (TMRE) (Figure 2(c)).
(c) Confocal fluorescence images of TMRE (C1: Ad-GFP, C2: Ad-UQCRC1, C3: Ad- UQCRC1+[Zn.sup.2+], and C4: AdUQCRC1+TPEN).
3a) that were labeled with MitoTracker Green FM (MTG FM; excited at 488 nm) and tetramethylrhodamine ethyl ester (TMRE
; excited at 561 nm).
Abbreviations: aer, alpha estrogen receptor; Bax, Bcl-2 like protein 4; Bcl-2, B-cell lymphoma 2; CFU, colony formation unit; dogp, octanol/water partition coefficient; COX-2, cyclooxigenase-2; DCFH-DA, dichloro-dihydro-fluorescein diacetate; [delta][psi]m, mitochondria potential; EDTA, Ethylenediamine-tetraacetic acid; EUG, eugenol; FITC, fluorescein isothiocyanate; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; [IC.sub.50], half maximal inhibitory concentration; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl-tetrazolium bromide; nfxb, nuclear factor kappa-light-chain-enhancer of activated B cells; OD, optical density; pcna, proliferation cell nuclear antigen; PI, propide iodide; ROS, reactive oxygen species; TMRE
, tetramethylrhodamine, ethyl ester.
To investigate the role of NF-[kappa]B pathway in preserving mitochondrial function in cardiomyocytes, TMRE
, a sensitive fluorescent probe that reflects the level of membrane potential, were used to assess the capacity of mitochondria to produce ATP.
After incubation, the amount of TMRE
fluorescence captured by each sperm mitochondrion was recorded by the software LAS AF Lite (Leica[R] Microsystems, Germany) at an emission of 500 nm and excitation of 600 nm by a fluorescence microscope (Leica Microsystems, Germany).
, a membrane-sensitive mitochondrial dye, was used to co-stain synaptic mitochondria (Fig.
To assess mitochondrial membrane potential, we used fluorescent dye TMRE
that is cell permeant, and it specifically accumulates in mitochondria with membrane potential.
 by measuring the extent of uptake of the potentiometric dye, tetramethyl rhodamine methyl ester (TMRE
, Sigma-Aldrich, Italy), normalized to MitoTracker Green (MTG), which is taken up by mitochondria independently of membrane potential.
Thereafter, cells were harvested, washed, and incubated for 30 min at 37[degrees]C in NaCl ringer solution (see above) containing 25 nM of the inner mitochondrial membrane potential ([DELTA][[PSI].sub.m]) specific dye tetramethylrhodamine ethyl ester perchlorate (TMRE
, Invitrogen) and [DELTA][[PSI].sub.m] was analyzed by flow cytometry in fluorescence channel FL-2 (logarithmic scale).