Apigenin (4′,5,7-trihydroxyflavone) with ≥ 98% purity was purchased from Cayman Chemical Company (Cat No. 10010275, Ann Arbor, MI, USA). Dimethyl sulfoxide (DMSO), diphenyl-1-pyrenylphosphine (DPPP), Dulbecco's modified Eagle's medium (DMEM), violet crystal, and tetramethylrhodamine ethyl ester (TMRE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), penicillin/streptomycin, trypsin/EDTA solution, and trypan blue were purchased from Gibco (Grand Island, NY, USA). Annexin V/FITC, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and propidium iodide were obtained from Invitrogen (Eugene, OR, USA). X-Gal was obtained from Life Technologies (Grand Island, NY, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) was obtained from Molecular Probes (Eugene, OR, USA). Matrigel was purchased from Becton–Dickinson (San Jose, CA, USA). All other reagents were purchased from Synth (Diadema, SP, BR).
Tetramethylrhodamine ethyl ester tmre
Manufactured by Merck Group
Sourced in United States, Germany
Tetramethylrhodamine ethyl ester (TMRE) is a fluorescent dye used for labeling and detecting mitochondria in living cells. It is a lipophilic cation that accumulates in active mitochondria due to the negative membrane potential. TMRE can be used to monitor mitochondrial membrane potential, an indicator of mitochondrial health and function.
Automatically generated - may contain errors
Lab products found in correlation
Mitogreen, by PromoCell (3 mentions)
Mitophagy detection kit, by Dojindo Laboratories (2 mentions)
All in one fluorescence microscope, by Keyence (2 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
X gal, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Dihydrorhodamine 123 dhr123, by Merck Group (1 mentions)
Bz x710, by Keyence (1 mentions)
Ethidium homodimer, by Merck Group (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Annexin 5 allophycocyanin ann 5 apc, by BD (1 mentions)
Qvd oph qvd, by Abcam (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
8 protocols using tetramethylrhodamine ethyl ester tmre
1
Apigenin Cytotoxicity and Oxidative Stress
2
Mitochondrial Assessment after GEM Treatment
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To evaluate mitochondrial status after GEM treatment, 2.0 × 104 cells were seeded in 12-well plates with or without GEM (0.1 μM), and each assay was performed after 72 h of incubation. The mitochondrial volume was assessed using MitoGreen (100 nM, for 15 min at 37 °C, PromoCell GmbH, Heidelberg, Germany). Mitochondrial potential was examined using tetramethylrhodamine ethyl ester (TMRE) (200 nM for 30 min at 37 °C, Sigma-Aldrich). Mitochondrial ROS levels were examined using dihydrorhodamine 123 (DHR123; 10 μM, 20 min at 37 °C; Sigma-Aldrich). Ethidium bromide (EtBr) was used to detect mtDNA and cytosolic EtBr accumulation was measured. Before imaging, cells were washed with Hank’s balanced salt solution. Fluorescence was measured using an all-in-one fluorescence microscope (BZ-X710; KEYENCE Co., Itasca, IL, USA). We used the NIH ImageJ software (version 1.52, Bethesda, MD, USA) to analyze the median fluorescence intensity per cell.
3
Hippocampal Neuron Hypoxia Assay
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As described above, hippocampal neurons were derived from E17–18 pups and incubated for seven days. The neurons were pretreated with QBA (0–30 µM) for 48 h and subsequently exposed to hypoxia (<0.3% O2) for 48 h. The cultures were stained with calcein AM (Invitrogen), a marker of live cells, with ethidium homodimer (Sigma-Aldrich), a marker of cell death, and with tetramethylrhodamine ethyl ester (TMRE; Sigma-Aldrich), a marker for mitochondrial health [27 (link)]. The fluorescence of each of these markers was then quantitated with a fluorimeter.
4
Mitochondrial Function Assessment Protocols
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Mitochondrial function was examined using fluorescent probes. After treatment with or without BBR (25 μM), cells were incubated with the probes for 30 min at 37 °C and photographed using an all-in-one fluorescence microscope (KEYENCE). We employed OxiORANGE (mtHR) (10 μM, Goryo Chemicals, Sapporo, Japan) to assess oxidative stress, mitoGreen (100 nM, PromoCell GmbH, Heidelberg, Germany) to assess mtVol, tetramethylrhodamine ethyl ester (TMRE) (200 nM, Sigma-Aldrich, St. Louis, MO, USA) to assess MMP, and the mitophagy Detection Kit (Dojindo, Kumamoto, Japan) as per the manufacturer’s instructions.
5
Apoptosis Quantification in Cells Treated with PKHB1
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Annexin‐V‐allophycocyanin (Ann‐V‐APC 0.1 μg/mL; BD Pharmingen, San Jose CA, USA), propidium iodide (PI, 0.5 μg/mL; Sigma‐Aldrich), and tetramethylrhodamine ethyl ester (TMRE, 20 nmol/L; Sigma‐Aldrich) were used for phosphatidylserine exposure, cell viability, and mitochondrial transmembrane potential (ΔΨm) quantification, respectively, in a BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) (total population 10 000 cells). Data were analyzed using FlowJo software (LLC, Ashland, OR, USA). Then, 1 × 106 cells/mL were treated for 2 hours with PKHB1 (as indicated). For the inhibition assays, calcium chelator, BAPTA (5 mmol/L, CalbioChem; Merck, Billerica, MA, USA) or the pan‐caspase inhibitor Q‐VD‐OPh (QVD, 10 μmol/L; BioVision, Milpitas, CA, USA) was added 30 minutes before PKHB1.
6
Mitochondrial Function Assessment Protocol
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Mitochondrial functions were examined using fluorescent probes. After treatment with or without BBR (25 μM), cells were incubated with the probes for 30 min at 37 °C and then photographed using an All-in-One fluorescence microscope (KEYENCE). We used MitoROS (mitochondrial superoxide) (10 μM, AAT Bioquest Inc., Sunnyvale, CA, USA) to assess oxidative stress, mitoGreen (100 nM, PromoCell GmbH, Heidelberg, Germany) to assess mitochondrial volume, tetramethylrhodamine ethyl ester (TMRE) (200 nM, Sigma-Aldrich) to assess MMP, and mitoFerrogreen (20 nM, Dojindo, Kumamoto, Japan) to assess mitochondrial iron (Fe2+). Mitophagy was detected using a Mitophagy Detection Kit (Dojindo), according to the manufacturer’s instructions.
7
In Vitro Cytotoxicity and Antioxidant Assays
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Methanol, ethanol, sulfuric acid (98%), trichloroacetic acid (≥ 98%), petroleum ether, anhydrous ammonia (≥99.95%), dichloromethane, chloroform, iron (III) chloride (FeCl 3 ), sodium sulphate (Na 2 SO 4 ), sodium citrate (Na 3 C 6 H 5 O 7 ), 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium (MTT), neutral red (NR), 2 ', 7'-dichlorodihydrofuorescein diacetate (DCFH-DA), tetramethylrhodamine ethyl ester (TMRE) all of analytical grade were obtained from Sigma Aldrich (St. Louis, MO, USA). Potassium hexacyanoferrate [K 3 Fe (CN) 6 ], 2,2-diphenyl-2-picryl-hydrazyl (DPPH), Ascorbic acid, β-carotene, linoleic acid, Triton X-100, Dimethyl sulfoxide (DMSO), Dulbecco's Modified Eagle's Medium (DMEM), metyrapone, quinidine and Hanks' Balanced Salt Solution (HBSS) of biochemical reagent grade were purchased from Merck Millipore (Darmstadt, Germany).
8
Mitochondrial Membrane Polarity Assessment
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Because loss of mitochondrial membrane polarity is an early marker of cardiomyocyte death (47), we determined whether myoblast-secreted mediators are able to preserve mitochondrial membrane polarity. Cardiac cell cultures were pretreated with MCM, CFCM, or fresh culture medium for 24 h before inducing oxidative stress with H 2 O 2 (100 µM) for another 24 h. Then cultures were treated with 100 nM tetramethylrhodamine ethyl ester (TMRE; Sigma-Aldrich) for 15 min and washed thereafter to remove any excess dye. TMRE diffuses to functional mitochondria where it exhibits bright fluorescence. This signal is quenched upon opening of the mitochondrial transition pore and the subsequent loss of mitochondrial membrane potential. Fluorescence signal was quantified with Wallac Victor2, and fluorescence images were acquired with an inverted fluorescent microscope (Olympus, Tokyo, Japan)
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