Each well was seeded with 1×105 Cal-27 cells. After 24 h of incubation, different concentrations of ICD were added (0, 0.15, 0.30, 0.60, and 1.20 mM) for 24 h, 48 h, and 72 h, respectively. After the cells were harvested, the levels and activities of the MMP (M8650, Solarbio, Beijing, China), ATP (BC0300, Solarbio, Beijing, China), ROS (CA1410, Solarbio, Beijing, China), and mitochondrial complex enzymes I–IV (BC0630; BC0950; BC3245; BC0945, Solarbio, Beijing, China) were detected according to the manufacturer’s instructions. The changes in the MMP in each group were detected with a 490 nm excitation wave and a 530 nm irradiation wave. The ATP content of each group was detected at a 340 nm wavelength. ROS levels were measured at a 488 nm excitation wavelength and a 525 nm emission wavelength. In addition, the cellular ROS content was calculated using flow cytometry (CytoFlex, Indianapolis, IN, USA) after 24 h, 48 h, and 72 h of treatment with 0.60 mM ICD, respectively. The detection wavelength of complex enzyme I was 600 nm, while that of complex enzyme II was 600 nm, that of complex enzyme III was 550 nm, and that of complex enzyme IV was 550 nm. The activity of the respiratory electron transport chain complex enzymes I–IV was detected in each group.
M8650
Manufactured by Solarbio
Sourced in China
The M8650 is a piece of laboratory equipment designed for the purpose of [core function]. It is a [brief description of key features and specifications].
Automatically generated - may contain errors
Lab products found in correlation
Ca1410, by Solarbio (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Bc0630, by Solarbio (1 mentions)
Bc0950, by Solarbio (1 mentions)
Bc0945, by Solarbio (1 mentions)
Bc3245, by Solarbio (1 mentions)
Bc0300, by Solarbio (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Ix73p2f, by Olympus (1 mentions)
A015 3, by Nanjing Jiancheng (1 mentions)
A001 3, by Nanjing Jiancheng (1 mentions)
Fluoview fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
Ti2 u, by Nikon (1 mentions)
Jc 1 kit, by Solarbio (1 mentions)
C2006, by Beyotime (1 mentions)
Cytoflex flow cytometry, by Beckman Coulter (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
17 protocols using m8650
1
ICD Effects on Cell Metabolism
2
Mitochondrial Membrane Potential Evaluation
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The mitochondrial membrane potential (MMP) was evaluated by JC-1 staining (M8650, Solarbio, Beijing, China). Cells were plated in 6-well plates and cultured in medium with or without mannose for 48 h. Cells were incubated for 30 min for mitochondrial staining with JC-1, and nuclei were then stained with Hoechst 33342 (100 nmol l−1; C1028, Beyotime, Shanghai, China) for 10 min. After staining, cells were observed under a confocal microscope (LSM880, Zeiss, Jena, Germany). JC-1 aggregates (red fluorescence) indicate a normal MMP, while JC-1 monomers (green fluorescence) indicate a decreased MMP.
3
Measuring Mitochondrial Membrane Potential
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An MMP detection kit (JC-1, M8650, Solarbio) was used to detect changes in MMP. After different treatments, RSCs were incubated with JC-1 staining working solution for 20 min in a 37°C incubator. The supernatant was removed by aspiration and washed twice with JC-1 staining buffer (1×). Two milliliters of DMEM medium was added to the six-well plate, and cells were imaged using an IX71 inverted fluorescence microscope.
4
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was assayed according to the manufacturer’s (M8650, Solarbio) instructions. First, the medium of the 6-well plate was removed and washed with PBS. Then, 1 ml cell culture medium and 1 mL JC-1 (1 ×) staining working solution were added to each well. The cells were incubated at 37°C for 30 min. After washing with JC-1 buffer, 1 mL medium was added to each well and observed under an inverted fluorescent microscope (OLYMPUS, IX73P2F).
5
Evaluating Mitochondrial Membrane Potential in hiPSC-CMs
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Changes in the mitochondrial membrane potential of different groups of hiPSC‐CMs were evaluated by JC‐1 staining (Solarbio®, M8650). hiPSC‐CMs were cultured on 96‐well collagen‐coated plates. Different groups of hiPSC‐CMs were washed with cold PBS and incubated with 100 μL JC‐1 work solution for 30 minutes at 37°C in the dark. The image was then scanned using a high‐content analyzer (Molecular Devices, USA), using the MetaXpress high‐content analysis system (ACEA Biosciences, USA) to set the cell parameters and automatically calculate the average fluorescence intensity of the cells.
6
Oxidative Stress and Mitochondrial Dysfunction Assessment
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ROS production was detected, based on the oxidation of 7-dichlorodihydrofluorescein diacetate (DCFH-DA) to 2,7-dichlorodihydrofluorescein, with a ROS detection kit (Boster, China) following manufacture’ instruction. DNA damage was observed via 8OHdG immunofluorescence staining. Mitochondrial membrane potential (MMP) in PMEVCs were detected by JC-1 staining (M8650, Solarbio, Beijing, China) according to the manufacturer's instructions. Culture medium supernatant were collected by centrifugation at 12,000×g for 5 min to measure antioxidant activity, including ferric ion reducing antioxidant power (FRAP) (A015-3, Nanjing Jiancheng Bioengineering Institute, China), GSH (A005, Nanjing Jiancheng Bioengineering Institute), or SOD (A001-3, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. Each determination was made the average of at least three independent experiments.
7
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential (ΔΨm) was measured by a JC-1 probe (M8650; Solarbio Science, Beijing, China) according to the manufacturer’s instructions. JC-1 located on mitochondria under normal conditions is shown in red, while JC-1 appears in green under cell depolarization. THP-1 macrophages treated as described above were stained by JC-1 and subsequently imaged by confocal laser scanning microscopy. The Image software was applied to evaluate fluorescence intensity, and the ratio of red/green indicated the degree of depolarization.
8
Mitochondrial Membrane Potential Assay
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Ishikawa cells were seeded in 6-well plates and cultured at 37 °C for 24 h, cells were treated with drugs for 12 h (NC group: complete culture medium; CuCy group: 0.5 μM CuCy; DSF group: 0.5 μM DSF; CuCy + DSF group: 0.5 μM CuCy + 0.5 μM DSF). Each group had three replicate wells, and each well contained 2 mL of culture medium. After discarding the culture medium, cells were washed once with PBS, followed by the addition of 1 mL of cell culture medium and 1 mL of JC-1 staining working solution (M8650, Solarbio). The mixture was thoroughly mixed and incubated at 37 °C in a cell culture incubator for 20 min. After the incubation period, the supernatant was removed, and cells were washed twice with JC-1 staining buffer (1x). Subsequently, 2 mL of cell culture medium was added, and the fluorescence was observed under a fluorescence microscope.
9
Monitoring Mitochondrial Membrane Potential in Hippocampal Neurons
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The changes in MMP of primary hippocampal neurons were monitored using JC-1 staining, a dual emission probe that exists as monomers with green fluorescence at low MMP and forms aggregates with red fluorescence at high MMP [81 (link)]. Primary hippocampal neurons were seeded and maintained in glass bottom dishes until 7 DIV and were stimulated with 1 nM NPCT or d-NPCT for another 24 h. After washing with warm HBSS three times, JC-1 (1:200; Solarbio Cat# M8650, Beijing, China) was diluted with NB, added back to the dishes, and incubated for 20 min at 37 °C and 5% CO2/95% air. The change of red and green fluorescence was monitored with a confocal microscope (Olympus Confocal Laser Scanning Microscope Fluoview FV3000, RRID:SCR_017015, Tokyo, Japan). The ratio of red/green fluorescence intensity was analysed using ImageJ software v.1.52a (National Institutes of Health, MD, USA; RRID:SCR_003070). The alterations of MMP in the in vitro SE model was monitored by the same JC-1 staining method, at the time points as those in the in vitro neuronal death assays.
10
Mitochondrial Membrane Potential Assay
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The mitochondrial membrane potential (MMP, ΔΨm) of cells was detected using the MMP probe JC-1 according to the protocol of the JC-1 kit (Solarbio, M8650). Briefly, cells were seeded in 24-well plates at 5 × 105 per well and treated with 10 μmol/L FIIN-2 for 24 h. After adding 0.5 ml F12 medium/JC-1 working solution (1:1), the cells were incubated at 37 °C for 20 min. The fluorescence signal was detected with a fluorescence microscope (Nikon, Ti2-U). The MMP of cells was represented by the ratio of aggregated JC-1 (red)/monomeric JC-1 (green).
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