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11 protocols using JC-1 Mitochondrial Membrane Potential Assay Kit

JC-1 assay was performed according to the manufacturer’s instructions (JC-1 Mitochondrial Membrane Potential Assay Kit; Abnova, Taipei City, Taiwan). Cells were cultured with different concentrations of ZnO NPs for 12 h under the conditions described above. They were then transferred onto a coverslip housed in a four-well plate and incubated in DMEM containing 10 μM JC-1 at 37°C for 15 min before washing with PBS and rapidly mounted for observation. In all procedures, cells were mounted with Vectashield fluorescence medium and visualized under a fluorescent microscope.
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Graphite flakes (109 meshes), sodium dodecyl sulfate, sulphuric acid (H2SO4 98%), phosphoric acid (H3PO4), potassium permanganate (KMnO4) and hydrogen peroxide were from Sigma Aldrich (St Louis, MO, USA) and were used without further purification. Hydrochloric acid (HCl, 37%), Diethyl ether, ethyl alcohol (99.7% v/v), sodium hydroxide, acetic acid glacial and 37% formaldehyde were from Friedemann Schmidt (Parkwood, WA, USA). Pristine protocatechuic acid (PCA) compound was purchased from Sigma Aldrich, USA. We also used the following: LDH test-kit (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega Co., Madison, WI, USA), Annexin V/FITC Apoptosis Detection Kit I (BD Biosciences, San Jose CA, USA), Propidium iodide (PI) Sigma-Aldrich, (St. Louis, MO, USA), CycleTESTTM PLUS DNA Reagent Kit (BD Biosciences, San Jose, CA, USA), JC-1 Mitochondrial Membrane Potential Assay Kit (Abnova, Middlesex County, NJ, USA), OxiSelect™ IntracellularROS Assay Kit (Green Fluorescence) (Cell BioLabs, Inc., San Diego, CA, USA). Phosphate Buffer Solution, methanol, and ethanol were all sourced from Sigma Aldrich (St. Louis, MO, USA).
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Graphite powder, sulphuric acid (H2SO4 98%), phosphoric acid (H3PO4), potassium permanganate (KMnO4), PEG4000, N-(3-Dimethylaminopropyl-N′-ethylcarbodiimide hydrochloride (EDC), 240 mg N-hydroxysulfosuccinimide (NHS), hydrogen peroxide, phosphate buffer solution, methanol, and ethanol were all sourced from Sigma Aldrich, St. Louis, MO, USA. Pristine protocatechuic acid (PA) and chlorogenic acid (CA) was purchased from Sigma Aldrich, USA. LDH test-kit (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega Co., Madison, WI, USA), Annexin V/FITC Apoptosis Detection Kit and CycleTEST TM PLUS DNA Reagent Kit from (BD Biosciences, San Jose, CA, USA), JC-1 Mitochondrial Membrane Potential Assay Kit (Abnova, Middlesex County, NJ, USA), OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) (Cell BioLabs, Inc., San Diego, CA, USA).
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The mitochondrial potential was quantified using the JC-1 mitochondrial membrane potential assay kit (Abnova, Taipé, Taiwan) according to manufacturer’s instructions and as previously described [18 (link)]. Briefly, 2 × 105 A2780 cells were seeded on each well of a 6-well plate and incubated for 48 h with IC50 JHOR11, 0.1% (v/v) DMSO or 3.5 µM cisplatin. Cells with red (JC-1 aggregated) and green (JC-1 monomer) fluorescence were quantified with an Attune acoustic focusing flow cytometer and respective software (Thermo Fisher Scientific), and fluorescence ratios normalized to control samples incubated with DMSO.
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5

Quantification of Apoptosis and Mitochondrial Membrane Potential in ZnO NP-Treated Cells

TUNEL analysis was performed to quantify cell apoptosis using the In Situ Cell Death Detection Kit, Fluorescein, purchased from Roche Diagnostics (Indianapolis, IN, USA) according to the manufacturer’s instructions. Cells were cultured with 15 μg/mL ZnO NPs for 0, 3, 6, and 12 hours, and then washed in PBS, fixed in 4% paraformaldehyde for 15 minutes, and incubated in a TUNEL reaction mixture for 2 hours at 37°C.
JC-1 assay was performed according to the manufacturer’s instructions (JC-1 Mitochondrial Membrane Potential Assay Kit; Abnova, Taipei City, Taiwan). Cells were cultured with 15 μg/mL ZnO NPs for 0, 3, 6, and 12 hours under the conditions described above. They were then transferred onto a coverslip housed in a four-well plate and incubated in DMEM containing 10 μM JC-1 at 37°C for 15 minutes before they were washed with PBS and rapidly mounted for observation. In all procedures, cells were mounted with Vectashield fluorescence medium and visualized under a fluorescent microscope.
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The mitochondrial membrane potential was assessed using a JC-1 Mitochondrial Membrane Potential Assay Kit (Abnova, Taipei, Taiwan) according to the manufacturer's instructions. In brief, cells in the fresh and cryogroups were cultured in 6-well plates (Thermo Fisher Scientific) in DMEM supplemented with 10% FBS at 39°C in a humidified incubator containing 5% CO2 and 5% O2. Cells were washed twice with DPBS and treated with JC-1 staining solution at 39°C for 15 min. To label nuclei, cells were counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The mitochondrial membrane potential was evaluated as the ratio between JC-1 aggregates (high mitochondrial membrane potential) and monomers (low mitochondrial membrane potential).
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To estimate changes in the mitochondrial membrane potential, a JC-1 Mitochondrial Membrane Potential Assay kit (Abnova) was used. Briefly, fibroblasts treated with various antioxidants after 22, 25, 28, and 31 passages (corresponding to about 31, 35, 39 and 43 PD) were seeded into 96-well flat clear-bottom black plates at a density 7.5 × 103/well and allowed to attach for 24 h at 37 °C. Then, 10 µL of JC-1 staining solution was added into the medium of each well and the plate was incubated at 37 °C for 30 min, then centrifuged at 4000 rpm for 5 min in an Eppendorf 5810R centrifuge and the supernatants were gently removed. The wells were washed twice using the buffer included in the kit, with centrifugation to avoid cell loss. The final supernatant was replaced by the buffer provided by the kit manufacturer (100 µL/well) and fluorescence was measured at 535/595 nm (JC-1 aggregates) and 485/535 nm (JC-1 monomers). The results are shown as a ratio of the fluorescence of JC-1 aggregates (red) to the fluorescence of JC-2 monomers (green).
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MSC were tested with the JC1-Mitochondrial Membrane Potential Assay Kit (Abnova, LuBioScience) which contains tetraethylbenzimidazolylcarbocyanine iodide (JC-1; LucernaChem, Lucerne, Switzerland), a cationic dye that accumulates in energized mitochondria. At low mitochondrial membrane potential JC-1 is predominantly a monomer (λex = 535 nm), while at high mitochondrial membrane potential the dye aggregates (λex = 595 nm). Cells were incubated for 15 min with JC-1 in growing medium, followed by three washes with PBS. Mitochondrial membrane potential was calculated as the ratio between JC-1 aggregate and monomer.
The production of superoxide by mitochondria was measured using the MitoSOX Red reagent (Thermo Fisher, Zug, Switzerland). Cells were incubated for 10 min with 5 μM MitoSOX Red and 1× SYBR (Thermo Fisher) in HBSS (Gibco), followed by three washes with HBSS. The oxidized product is highly fluorescent upon binding to nucleic acid. Bottom well fluorescence absorbance was measured using a Multimode Detector for both methods and results were normalized on RFU of SYBR.
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NSCs were seeded at a density of 5 × 105/ml per well of a 96-well culture plate. Cells were exposed to OGD with AC (0.1 and 1 µM) and incubated for 8 h at 37 °C. Mitochondrial membrane potential was measured using a JC-1 Mitochondrial Membrane Potential Assay Kit (Abnova, Taipei Taiwan) according to the manufacturer’s instructions. Mitochondrial membrane potential was assessed using an ELISA plate reader (Synergy H1 reader, BioTek Instruments). Apoptotic or unhealthy cells with mainly JC-1 monomers were detected with settings designed to detect FITC (excitation/emission = 485/535 nm).
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JC-1 assay was performed according to the manufacturer’s instructions (JC-1 Mitochondrial Membrane Potential Assay Kit; Abnova, Taipei, Taiwan). SKOV3 cells were plated in six-well plates (2×105 cells per well) and incubated with rGO-Ag (0.2 µM) or TSA (0.2 µM) or combination of rGO-Ag (0.2 µM) and TSA (0.2 µM) for 24 h under the conditions described above. The cells were then transferred onto a coverslip housed in a four-well plate and incubated in DMEM containing 10 µM JC-1 at 37°C for 15 min before washing with PBS and were rapidly mounted for observation. In all procedures, cells were mounted with Vectachield fluorescent medium and visualized under a fluorescent microscope.
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