For comparing mitochondrial mass and activity, islets from 2–3 month-old MNYI mice (both males and females) were isolated and dissociated into single cells. They were then stained with MitoView™ 633 or MitoView™ 650 (Biotium) at 37 °C for 15 minutes in the presence of 20 mM glucose, followed by Flow-cytometry analysis (Ernst et al., 2023 (link)). These dyes assay the mitochondrial transmembrane potential and mitochondrial mass, respectively. The β-cells were gated into eYFP+ and eYFP− β-cell subtypes in order to compare their MitoView™ signals. For ADP/ATP ratio in purified β-cell subtypes, an EnzyLight™ ADP/ATP ratio Assay Kit (BioAssay Syetems) was used, following the manufacturer’s protocols.
Mitoview 633
Manufactured by Biotium
Sourced in United States
MitoView 633 is a fluorescent mitochondrial stain that specifically labels mitochondria in live cells. It is a far-red dye that can be detected using the Cy5 or similar optical filters.
Automatically generated - may contain errors
Lab products found in correlation
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Mitoview 650, by Biotium (1 mentions)
Rhodamine 800, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mitotracker green fm probe, by Thermo Fisher Scientific (1 mentions)
Sytox blue dead stain, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Ldh cytotoxicity assay kit, by Promega (1 mentions)
Sh sy5y cell, by American Type Culture Collection (1 mentions)
Caspase 3 antibody 9662, by Cell Signaling Technology (1 mentions)
Bcl xl, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Tetramethylrhodamine methyl ester tmrm, by Thermo Fisher Scientific (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Cellrox green, by Thermo Fisher Scientific (1 mentions)
Cellrox orange, by Thermo Fisher Scientific (1 mentions)
To pro 3, by Thermo Fisher Scientific (1 mentions)
13 protocols using mitoview 633
1
Mitochondrial Profiling in Beta-Cell Subtypes
2
Mitochondrial Labeling with Photosensitizers
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Mitotracker Deep Red FM (Life Technologies, #M22426), Mitoview 633 (Biotium, #70055), Rhodamine 800 (Sigma, #83701), and TMRE (Life Technologies, #T-669) were used without further purification. Photosensitizer stock solutions were prepared in DMSO (Sigma, #D2650). Absorption spectra of the photosensitizers were determined on a UV-Vis spectrophotometer (Beckman Coulter, #DU-800) in PBS buffer (pH 7.4) at room temperature within a quartz cuvette (Nova Biotech, #QS-467). Condition for labeling HeLa cell mitochondria with photosensitizers: 100 nM in complete medium at 37 oC for 30 min (followed by 3X PBS wash).
3
Apoptosis Assay of Keratinocytes
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Keratinocytes infected with control, NUP98, or RAE1 shRNA were seeded onto 24-well plate. A positive control was made by adding H2O2 (2 mM) to keratinocytes expressing control shRNA for 6 h at 37 °C. MitoView 633 (Biotium) and Aquaphile JC1 (Biotium) apoptosis assay kits were used to assess induction of apoptosis with loss of NUP98 or RAE1. JC1 or Mitoview was added at 1:1000 in keratinocyte culture medium, and incubated for 20 min in the CO2 incubator. Hoechst was added at 10 μg/mL for 5 min to stain the DNA before image acquisition.
4
Mitochondrial Function in Primary Monocytes
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Primary monocytes were cultured and treated as indicated. Thirty minutes before harvest, cells were incubated at 37 °C with 100 nM MitoTracker Green FM probe (Life Technologies) and 100 nM MitoView 633 (Biotium) for 30 m in dark to evaluate mitochondrial mass and ΔΨm, respectively. After harvest, cells were stained with SYTOX Blue dead stain (Life Technologies) as recommened by vendor to monitor cell viability. Data were acquired on a BD LSRFortessa (BD Biosciences). Analysis of data was accomplished as indicated above.
5
Molecular Apoptosis Mechanisms in SH‐SY5Y Cells
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SH‐SY5Y cells were obtained from ATCC. All cells were cultured in DMEM supplemented with 10% FBS and non‐essential amino acids at 37°C in a humidified incubator containing 5% CO2. The BCL‐2, BCL‐xL, AIF and BAX antibodies were obtained from Abcam. Caspase‐3 antibody (9662S) was obtained from Cell Signaling Technology. GAPDH and β‐actin antibodies were obtained from Proteintech. The LDH cytotoxicity assay kit (J2380) was obtained from Promega. ATP Cell Titer‐Glo assay kit was obtained from Promega. The Mitoview 633 and DAPI were obtained from Biotium.
6
Multiparametric Imaging of Cellular Homeostasis
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Imaging was performed by the following fluorescent dyes using the recommended doses according to the respective manufacturers: Fluo8 AM (2 μM; AAT Bioquest, Sunnyvale, CA, USA) to indicate calcium ions; MitoTracker Deep Red (1 μM; Life Technologies, Eugene, OR, USA) and MitoView 633 (1 μM; Biotium, Fremont, CA, USA) to label mitochondria; and tetramethylrhodamine methyl ester (TMRM, 200 nM; Life Technologies) to indicate mitochondrial membrane potential. To detect ROS, we used CellRox Green (CRG, 2 μM), CellRox Orange (CRO, 2 μM) and H2DCFDA (1 μM; all from Life Technologies). TO-PRO3 (100 nM; Life Technologies) was used as an early indicator of cell death. These dyes were applied to cells grown on glass-bottom dishes and incubated for 30 min in 37 °C in a CO2 incubator, followed by washing (×3 with imaging medium) before imaging.
7
Measuring Mitochondrial Membrane Potential
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Differences in membrane potential, were assessed using MitoView™ 633 (Biotium, Inc., California, USA) following manufacturer’s instructions. Briefly, after incubation with the dye for 1 h, fluorescence was measured in a microwell plate fluorescence reader, Infinite 200 PRO NanoQuant (TECAN).
8
Mitochondrial Staining in Keratinocytes
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Keratinocytes infected with either control or NUP93 shRNA were seeded onto a 24-well plate. Hydrogen peroxide (2 mM) was added to control knockdown keratinocytes for 6 h at 37 °C as a positive control. The Mitoview 633 (Biotium, 70055 T) reagent was reconstituted in DMSO according to manufacturer’s instructions and added at 1:1,000 and incubated for 20 min in the CO2 incubator. Prior to imaging, the DNA stain Hoechst was added at 10 ug/mL for 5 min.
9
Mitochondrial and Nucleolar Analysis in Cells
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Confocal images were acquired using LSM 780 confocal laser scanning microscope (Zeiss, Germany) with 405/488/561/633 nm lasers, processed with ZEN software (Zeiss), and analyzed by ImageJ/Fiji software (NIH). Live imaging was performed using confocal dish (200350; SPL Life Sciences, Korea) in a live cell chamber maintaining 5% CO2 at 37°C. Mitochondrial membrane potential was visualized by Mitoview633 (70055; Biotium, USA) fluorescent dye staining. The nuclear to cytoplasmic ratio of S-tdT, puromycylation, and RAN expression was calculated in individual cells by measuring their intensities of nuclear and cytoplasmic fluorescence as described previously (Lee et al., 2020 (link)). Mitochondrial morphology was analyzed using the Mitochondrial Analyzer plugin in ImageJ/Fiji (https://github.com/AhsenChaudhry/Mitochondria-Analyzer ) (Chaudhry et al., 2020 (link)). Nucleolar HSP70 was detected using Stardist plugin in Fiji (https://github.com/stardist/stardist/ ) (Schmidt et al., 2018 (link)).
10
Apoptosis detection in live keratinocytes
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MitoView 633 (Biotium) staining was used to determine potential induction of apoptosis in live keratinocytes. Keratinocytes were seeded onto a 24-well plate. A positive control was included by treating keratinocytes expressing control shRNA with H2O2 (2 mM) for 6 h at 37°C. MitoView was added to the keratinocytes at 1:000 and incubated for 20 min in the cell culture incubator. Hoechst 33342 (10 μg/mL, Thermo) was then added for 5 min to stain the DNA before imaging.
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