Mitochondrial content was measured using MitoTracker® Mitochondrion‐Selective Probes (Molecular Probes®). Cells were stained with a 20 nmol/L of MitoTracker Deep‐Red and incubated at 37°C 5% CO2/O2 for 60 min. Cells were then washed three times with sterile PBS and imaged in phenol red‐free FluoroBrite DMEM. MitoTracker® excitation and emission were in the red spectrum at 635/670 nm, respectively.
Mitotracker mitochondrion selective probe
Manufactured by Thermo Fisher Scientific
Sourced in United States
MitoTracker® Mitochondrion-Selective Probes are a series of fluorescent dyes that selectively stain mitochondria in live cells. They accumulate in active mitochondria and their fluorescence is dependent on membrane potential.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Mitotracker red cmxros m7512, by Thermo Fisher Scientific (1 mentions)
Rotenone, by Merck Group (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Z vad fmk, by R&D Systems (1 mentions)
Mdivi, by Merck Group (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Rnasin ribonuclease inhibitor, by Promega (1 mentions)
Rabbit cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Brilliant 3 ultra fast sybr green qpcr master mix, by Agilent Technologies (1 mentions)
12 protocols using mitotracker mitochondrion selective probe
1
Mitochondrial Content Quantification
2
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was measured in bone marrow cells and splenocytes of mice (isolated immediately after the sacrifice) using MitoTracker® Mitochondrion-Selective Probes (Molecular Probes, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), a green-fluorescent mitochondrial stain, which appears to localize to mitochondria regardless of mitochondrial membrane potential. To label mitochondria, live cells were incubated with 100 nM of MitoTracker® probes, which passively diffused across the plasma membrane and accumulated in active mitochondria. The reduced probes do not fluoresce until they enter live cells, where they are oxidized to the corresponding fluorescent mitochondrion-selective probe and then sequestered in the mitochondria. Suspension cells were centrifuged to obtain a cell pellet that was resuspended in a prewarmed (37 °C) MitoTracker® probe staining solution. After 30 min of incubation at 37 °C and 5% CO2, the cells were re-pelleted by centrifugation and resuspended in a fresh prewarmed medium or buffer. Green fluorescence was read at Ex/Em = 490/516 nm in a microplate reader. Data are expressed as M.F.I. (a.u.).
3
Mitochondrial Content Quantification via MitoTracker
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Mitochondrial content was measured using MitoTracker® Mitochondrion‐Selective Probes (Molecular Probes ®). Cells were stained with a 20 nmol/L of MitoTracker Deep‐Red and incubated at 37°C 5% CO2/O2 for 60 min. Cells were then, washed three times with sterile PBS and imaged in phenol red‐free FluoroBrite DMEM. MitoTracker® excitation and emission were in the red spectrum at 635/670 nm (Miyake et al. 2011 ; Jimenez et al. 2014 ).
4
Mitochondrial Content Quantification
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Mitochondrial content was measured using MitoTracker® Mitochondrion-Selective Probes (Molecular Probes® Cat No. M22426). Cells were stained with a 20 nM of MitoTracker Deep-Red and incubated at 37 °C, 5% CO2/O2 for 60 min. Cells were, then, washed three times with sterile PBS and imaged in phenol red-free FluoroBrite DMEM. MitoTracker® excitation and emission were in the red spectrum at 635/670 nm [37 ].
5
Visualizing Mitochondrial Dynamics in GSIV-Infected Cells
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GSM cells were cultured in confocal microscopy wells and transfected with pDsRed-Monomer-N1-Bcl-xL and pDsRed-Monomer-N1, respectively. After infection with GSIV (MOI = 0.5) for 24 h, the cells were stained with MitoTracker® mitochondrion-selective probes (Invitrogen) for 30 min at the culture temperature in the dark. The cells were then washed three times with PBS and fixed using 4% formaldehyde. The cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (10 µg/mL) (Solarbio, Beijing, China) for 10 min at room temperature in the dark. After washing three times with PBS, the cells were observed under a confocal microscope (Olympus).
6
Mitochondrial Imaging of Apoptosis
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Cells were seeded in 4-well plates, and then treated with DMSO or ABT-737 for 24 hr. To label mitochondria, cells were incubated with 500 nM MitoTracker® Mitochondrion-Selective Probes (Invitrogen, Carlsbad, CA, USA) at 37°C for 45 min, which passively diffuse across the plasma membrane and accumulate in active mitochondria. After staining live cells with a MitoTracker probes, cells were fixed and permeabilized using a Cytofix/Cytoperm Solution at 4°C for 1 hr. Cells were blocked with 1% BSA at RT for 1 hr and incubated at 4°C overnight, followed by incubation with FITC-conjugated secondary antibody at RT for 1 hr. Cells were observed by fluorescence microscopy with the appropriate filters for fluorescence dye.
7
Mitochondrial Staining of Fungal Conidia
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Five to eight 7-mm fungal plugs from the leading edge of a 3-d-old colony of the complemented strain (cFpppr1) on PDA were transferred into a 250 mL flask containing 100 mL of CMC liquid media with shaking at 150 rpm at 25°C for 4–5 d. The conidia were harvested into a 50 mL microcentrifuge tube after filtering through one-layer sterile Miracloth. The conidia suspension was centrifuged at 2,500 g and supernatant was removed then resuspended in 1 mL of Phosphate buffered saline (PBS) and centrifuged again. After the second wash, 1 × 105 conidia in a fresh tube were resuspended in prewarmed Mito-Tracker Red CMXRos M7512 at 37°C (MitoTracker mitochondrion-selective probes, Invitrogen) working solution (200 nM in PBS from the stock solution at −20°C, protected from light) for 10 min of staining following the kit instruction. The conidia were pelleted at 5,000 g for 3 min and resuspended in 50 μL of PBS for imaging with fluorescent GFPHQ green and TRITC red filters with a Nikon fluorescence Eclipse inverted Ti-S microscope.
8
Mitochondrial Localization in Oocytes
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Oocytes mitochondrial distribution was detected using MitoTracker® Mitochondrion-Selective Probes (M22426, Invitrogen, CA). According to the manufacturer's instructions, the probe was adjusted to the working solution concentration of 200 nM and then incubated. The oocyte samples were transferred to the working solution and incubated at 37°C for 30 min in the dark. After washing 3 times, it was transferred to a glass slide and images were taken using a fluorescence inverted microscope.
9
Mitochondrial Dynamics and Cell Death
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BRD, Mdivi, staurosporine, and rotenone were purchased from Sigma-Aldrich. Z-VAD FMK was purchased from R&D systems. MitoTracker Mitochondrion-Selective Probes were purchased from Invitrogen. Trizol was purchased from Life Technologies. M-MLV Reverse Transcriptase and RNasin Ribonuclease Inhibitor were purchased from Promega. Oligo(dT) primer was purchased from Fermentas Life Sciences and PCR Nucleotide Mix was purchased from GE Healthcare. Brilliant III Ultra-Fast SYBR Green QPCR Master Mix was purchased from Agilent Technologies. α/β-Tubulin rabbit, cleaved caspase-3 rabbit, DRP1 rabbit, phospho-DRP1 (Ser616) rabbit, HSP90 Rabbit, HMGB1 Rabbit, COX IV Rabbit, and anti-rabbit IgG HRP-linked antibodies were purchased from Cell Signaling Technology. Nlrx1 polyclonal antibody was purchased from Proteintech.
10
Mitochondrial Morphology Visualization
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MitoTracker® Mitochondrion-Selective Probe (Invitrogen, Gaithersburg, MD) was used to detect the morphology of mitochondria. Cells were incubated with the staining solution containing MitoTracker® probe (100 nM) at 37°C for 30 min and fixed with 4% formaldehyde, followed by DAPI staining. The images of each well were obtained in three different fields by confocal microscopy. The morphology of mitochondria was analyzed using Image J with mitochondrial network analysis toolset (MiNA).
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