The mitochondrial membrane potential (ΔΨ m ) was evaluated using 3,3`-dihexyloxacarbocyanine iodide [DiOC 6 (3)] (Sigma-Aldrich). DiOC 6 (3) is a cell-permeant, green fluorescent, lipophilic probe that is selective for mitochondria of live cells (Franklin et al., 2001) (link). Algal cells (1 × 10 6 mL -1 ) in OECD medium were stained with 2.5 μmol L -1 DiOC 6 (3) for 10 min at room temperature in the dark, as previously described (Sousa et al., 2018) (link). As negative control, cells were treated with 50 μmol L -1 of carbonyl cyanide m-chlorophenyl hydrazine (CCCP, Sigma-Aldrich) (an uncoupler of the proton gradient) for 10 min and then stained with DiOC 6 (3) as described above. CCCP stock solution (5 mmol L -1 ) was prepared in dimethyl sulfoxide (DMSO); solvent concentration in the negative control ≤ 1% (v/v). Fluorescence was quantified, in quintuplicate, as described in the metabolic activity assessment.
Dioc6 3
Manufactured by Merck Group
Sourced in United States, France
DiOC6(3) is a fluorescent dye used for the detection and analysis of mitochondrial membrane potential in cells. It is a cationic lipophilic dye that accumulates in the mitochondria of living cells in proportion to the membrane potential.
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17 protocols using dioc6 3
1
Mitochondrial Membrane Potential Analysis
2
Evaluation of Antidepressant-Induced Apoptosis
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Cells were treated with the indicated concentration of sertraline and paroxetine for 1.5, 3, 6, 12, and 24 h. Before being harvested, cells were incubated with 40 nM DiOC6(3) (Sigma) for 30 min at 37°C. After that, cells were washed and suspended in phosphate-buffered saline (PBS). DiOC6(3) fluorescence intensities of FL-1 (530±15 nm) were measured with a flow cytometer CellQuest program (FACScan, Becton Dickinson).
3
Mitochondrial Depolarization in Platelets
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For measuring Δψm depolarization, platelets were incubated with DiOC6(3), a cell-penetrating green-fluorescing dye, and analyzed by flow cytometry. Depolarization results in a reduced DiOC6(3) accumulation in mitochondria when Δψm is dissipated. PRP aliquots were treated with, PBS (negative-control), 10 μM ADP, 5 U/mL thrombin (Sigma-Aldrich) or leptospires, as described before, with the addition of 100 nM DiOC6(3) (Sigma-Aldrich). The samples were diluted to 500 μL with PBS and flow cytometric acquisition was performed. Δψm depolarization was quantified as a decrease of the mean channel fluorescence (MCF) of platelet-bound DiOC6(3) (CD41a-positive gate). Two independent experiments were performed, in triplicates. One-way ANOVA statistical analysis was performed (GraphPad Prism 8).
4
Fluoxetine Treatment and Mitochondrial Potential
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5
Preparation and Characterization of Crystalline CAP-NO
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The preparation and physicochemical characterization of crystalline CAP-NO were described previously41 (link)47 (link). ODQ and Hb were purchased from Sigma (St. Louis, MO). Human interleukin-1 beta (IL-1β), human interferon gama (IFN-γ), human tumor necrosis factor alpha (TNF-α), human lymphotoxin (LT, TNF-β) and mouse anti-human beta-actin (β-actin) antibody were purchased from Cell Signaling Technology, Inc. Recombinant human interleukin-6 (IL-6) was purchased from Peprotech, Inc. Mouse anti-human CD144-PE (P-phycoerythrin), mouse anti-human CD31-FITC (fluorescein isothiocyanate), mouse anti-human CD54-PE, mouse anti-human CD62E-APC (allophycocyanin), mouse anti-human CD29 (integrin β1)-PE, PE mouse IgG1 kappa isotype control, FITC mouse IgG1 kappa isotype control and APC mouse IgG1 kappa isotype control antibody were obtained from Becton Dickinson (BD) Pharmingen™. Mouse anti-human CD106-PE was obtained from eBioscience. Fibronectin and ECGS were obtained from BD Biocoat™. Rabbit anti-human VCAM-1 antibody was obtained from Abcam. Fluorescent dyes propidium iodide (PI) and DiOC6 (3) were obtained from Sigma-Aldrich. Protocols involving animal and human samples were approved by the Institute Animal Care and Use Committees in compliance with USDA regulations (National Research Council 1996).
6
Mitochondrial Membrane Potential Assay
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The cells were washed three times with PBS after 96 h of co-incubation with OCP and OCP-Sr, then detached from the surface of the culture plastic using 0.05% trypsin-EDTA solution and stained with 10 nM 3,3′-dihexyloxacarbocyanine iodide (DiOC6 (3)) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min in a CO2 incubator to measure mitochondrial membrane potential (ΔΨm). As a control, cells were incubated with 250 nM valinomycin (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The measurement was conducted by BD Accuri C6 flow cytometer. 3 × 104 cells were analyzed for each sample.
7
Mitochondrial Membrane Potential Assay
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The mitochondria membrane potential (ΔΨm) was measured by monitoring the fluorescence of the specific probe 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3); Sigma-Aldrich, Saint-Quentin Fallavier, France) as described by Tungmunnithum et al. [65 (link)]. At least six independent measurements were performed for each condition and the results were expressed as relative fluorescent units.
8
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane energization was determined by a lipophilic cationic fluorochrome dye, 3,3′-dihexyloxacarbocyanine iodide [DiOC6 (3)] method (Sigma-Aldrich Co).
Briefly, hMSCs and NCs were treated with 40 nM DiOC6(3) in culture medium and then incubated for 30 minutes at 37 °C in the dark. Cells were collected and washed in cold PBS. The fluorescent mean intensity was analysed using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 501 nm.
Briefly, hMSCs and NCs were treated with 40 nM DiOC6(3) in culture medium and then incubated for 30 minutes at 37 °C in the dark. Cells were collected and washed in cold PBS. The fluorescent mean intensity was analysed using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 501 nm.
9
Mitochondrial Membrane Potential in HepG2 Cells
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The MMP (ΔΨm) of HepG2 cells treated with green-synthesized ZnONPs was measured with 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3), Sigma) which works by staining mitochondria according to their MMP. Cells were incubated in culture media supplied with 25 nM DiOC6(3) at 37 °C for 40 min. MMP was expressed as relative fluorescent units (RFU). All the experiments were performed three times.
10
Assessing Mitochondrial Membrane Potential
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The mitochondrial transmembrane potential was measured by cytofluorimetry, using the fluorescent dye 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3), Sigma Aldrich]. DiOC6(3) is a green fluorescent membrane dye that has been used to detect mitochondrial membrane potential in live cells. The fluorescence of the dye is enhanced when incorporated into mitochondrial membranes. Changes in mitochondrial membrane potential were assessed in cells by measuring fluorescence intensity due to a conformational change of the dye. The G93ADOXY− cells, G93ADOXY+, and G93ADOXY+ treated with exosomes (1 × 106) were processed as described above. Cells were resuspended in PBS containing DiOC6(3) (1 nM), incubated at 37°C for 15 min, immediately analyzed by flow cytometry with a FACSCanto II (BD) and analyzed with FlowJo software (Treestar Inc.).
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