For mitochondrial ROS and membrane potential assays, neurons were grown and maintained as described above except that coverslips were pre-coated with 100 μg/mL poly-D-lysine. Neurons were 1) pretreated with 1 μM CAT-SKL for 24 hours, and post-treated with 1 μM ADDL for an additional 24 hours, or 2) pretreated with 1 μM ADDL for 24 hours and then post-treated with 1 μM CAT-SKL for an additional 24 hours. Mitochondrial ROS production was determined by using MitoTracker® Red CM-H2XRos (Invitrogen). Medium containing 200 nM of the Mitotracker probe was incubated with neurons for 20 minutes at 37°C, 5% CO2, and washed with Hank's balanced salt solution. Mitochondrial membrane potential was determined using the JC-1 mitochondrial potential sensor (Invitrogen). Depolarized mitochondria appear as diffuse green structures. Medium containing 2 μg/mL of the JC-1 dye was incubated with the neurons for 20 minutes, and washed with Hank's balanced salt solution. Cells from both treatment protocols were imaged using the Zeiss ApoTome Imaging system from the MICR.
Jc 1 mitochondrial potential sensor
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
The JC-1 mitochondrial potential sensor is a fluorescent probe used to measure mitochondrial membrane potential in cells. It exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (monomeric form) to red (aggregate form). This property allows for the detection of changes in mitochondrial potential, which is a widely used indicator of mitochondrial function.
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Lab products found in correlation
Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions)
Kaluza flow cytometry analysis software, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Cell culture plates, by Agilent Technologies (1 mentions)
Camptothecin cpt, by Merck Group (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
Cell clock cell cycle assay, by Biocolor (1 mentions)
6 protocols using jc 1 mitochondrial potential sensor
1
Mitochondrial ROS and Membrane Potential
2
Evaluating Mitochondrial Potential with JC-1
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The standard commercial protocol was followed for Invitrogen’s JC-1 mitochondrial potential sensor. In short, 2.0 μg/mL of JC-1 was diluted in 2% FBS-containing MMC media. This was then added to MMC cultures and incubated at 37°C for 20 min. Following this incubation, the cultures were gently triple-washed with PBS. Immediately after the washes, images were captured on the Keyence microscope before cells dried out. The ratio of red to green channel feedback was calculated based on the image analyzer’s results.
3
Mitochondrial Membrane Potential Quantification
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The mitochondrial membrane potential was examined using the JC-1mitochondrial potential sensor (Invitrogen, Carlsbad, CA). After treatment with IS and PCS, cells were incubated with the JC-1 dye (10 μg/mL) at 37°C for 15 min, followed by analysis with the Gallios flow cytometer (Beckman Coulter, Brea, CA) for quantifying 488-nm-excited fluorescence signals at 585/42 nm (FL2; red) and 525/50 nm (FL1; green). JC-1 monomers emit at 530 ±15 nm (FL1 channel), and J-aggregates emit at 590 ± 17.5 nm (FL2 channel). Cytometry settings were optimized for green (FL1) and red (FL2) fluorescence, and the data were analyzed with the Kaluza Flow Cytometry analysis software V1.2 (Beckman Coulter). The relative red to green fluorescence ratio of cells was calculated.
4
Podocyte Metabolic Function Analysis
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The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of podocytes were assessed by an Agilent Seahorse XF extracellular flux analyzer (Agilent Technologies) according to our previous description (16) . Briefly, podocytes were seeded into cell culture plates (Agilent Technologies). Data were calculated from three independent measurements obtained prior to or after compound injection.
MMP and NAD + Content Measurements MMP was measured using the JC-1 mitochondrial potential sensor (Invitrogen) according to the manufacturer's protocols. Podocytes were harvested and incubated with JC-1 solution, and the fluorescence of JC-1 monomers and J-aggregates was analyzed by flow cytometry. NAD 1 content was measured using an NAD 1 Assay Kit (Beyotime Biotechnology) according to the manufacturer's recommendation.
MMP and NAD + Content Measurements MMP was measured using the JC-1 mitochondrial potential sensor (Invitrogen) according to the manufacturer's protocols. Podocytes were harvested and incubated with JC-1 solution, and the fluorescence of JC-1 monomers and J-aggregates was analyzed by flow cytometry. NAD 1 content was measured using an NAD 1 Assay Kit (Beyotime Biotechnology) according to the manufacturer's recommendation.
5
Mitochondrial Membrane Potential Assay
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Astrocytes were plated onto a100mm culture dish at a density of 0.6 × 105/cm2 in astrocyte media. After 24 h the astrocytic conditioned media (ACM) was collected, and the mitochondria depleted ACM (dmACM) was obtained by passing the ACM through the 0.22 μm sterile filter. The mitochondrial membrane potentials were measured by the JC-1 mitochondrial potential sensors (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~ 525 nm) to red (~ 590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. These characteristics make JC-1 a sensitive marker for mitochondrial membrane potential. JC-1 dye (5 μM, 2 μg/ml) was added to ACM or dmACM and incubated for 30 min at room temperature before assaying. Mitochondria membrane potential was determined by the fluorescent ratio with a fluorescent microplate reader.
6
Investigating Anticancer Drug Effects
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NTD (2,3-dimethoxy-12-methyl-(1,3)-benzodioxolo(5,6-c)phenanthridinium) was used, as prepared in a previous report [6 (link)]. Camptothecin (CPT), topotecan (TPT), and paclitaxel (PTX) were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan). JC-1 mitochondrial potential sensors were purchased from Thermo Fisher Scientific K.K. (Kanagawa, Japan). A Cell Cycle Assay Cell-Clock was purchased from Biocolor Ltd. (County Antrim, UK).
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