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To test the hypotheses that these structural changes would be accompanied by similar level of mitochondrial maturation, we performed live staining of undifferentiated hESC (mTmG RuES2) and hESC-CM at early and late stages with two mitochondrial indicators, TMRM and Mito Tracker Green FM (MTG).
MMP levels were measured in cells treated with 30 nM tetramethylrhodamine (TMRM) for 30 min.
Abbreviations CT: Computed tomography DiO[C.sub.6]: 3,3-Dihexyloxacarbocyanine iodide DNA: Deoxyribonucleic acid GFP: Green fluorescent protein HIFU: High-intensity focused ultrasound JC-1: 5,5;,6,6-Tetrachloro-1,1 ;,3,3-tetraethylbenzimida zolylcarbocyanine iodide LITT: Laser interstitial thermal therapy MRI: Magnetic resonance imaging MW: Microwave RFA: Radiofrequency ablation [R.sub.m]: Cell membrane resistance TMRM: Tetramethylrhodamine.
Loss of mitochondrial membrane potential, an indicator of cellular apoptosis, was checked using tetramethylrhodamine methyl ester (TMRM), a cell permeant cationic red fluorescent dye (Griffin et al.
(17.) Scatena LM, Villa TCS, Ruffino Neto A, Kritski AL, Figueiredo TMRM, Vendramini SHF, et al.
Light in the ultraviolet (390/18), cyan (475/28) and green (549/15) channels was used to excite Hoechst-33342, GFP and TMRM, respectively.
For the assessment of mitochondrial transmembrane potential, cells were incubated with a 50 nM mitochondria-specific dye, TMRM, in medium for 15 minutes at 37[degrees]C, followed by two gentle washes with PBS and immediately analyzed by flow cytometry.
For other stainings, MitoTracker Green FM was added to live, unfixed HepaRG cells at a concentration of 0.1 [micro]M and incubated for 30 minutes prior to imaging; mBCI dye was added to live, unfixed HepaRG cells at a concentration of 80 [micro]M and incubated for 10 minutes prior to imaging; CellTracker Red CMTPX was added to live, unfixed HepaRG cells at a concentration of 0.5 [micro]M and incubated for 30 minutes prior to imaging; TMRM dye was added to live, unfixed HepaRG cells at a concentration of 0.1 [micro]M and incubated for 30 minutes prior to imaging; Alexa Fluor 568 Phalloidin was added to fixed HepaRG cells at a concentration of 0.165 [micro]M and incubated for 60 minutes prior to imaging.
All of these apply, however, to other nonratiometric [DELTA][PSI]m indicators (e.g., rhodamine 123 or TMRM), and it may markedly complicate data analyses, especially when these compounds are used in low-resolution approaches, such as flow-cytometry and cuvette-based spectrophotometric assays.
(31.) Scatena LM, Villa TCS, Ruffino Netto A, Kritski AL, Figueiredo TMRM, Vendramini SHF, Assis MMA, Motta MCS.
(5.) Figueiredo TMRM. Acesso ao tratamento de tuberculose: avaliacao das caracteristicas organizacionais e de desempenho dos servicos de saude--Campina Grande/ PB, Brasil [tese de doutorado].
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