Cultures of wild-type BY4741 S. cerevisiae grown in YPD overnight were passaged to fresh YPD to OD600 0.3–0.5 and grown another 4–5 hours. The culture was divided and dH2O (no treatment), 1% DMSO or the indicated concentrations of fludioxonil (in 1% DMSO) was added to each aliquot. Cells were grown for 30 minutes, after which 100 nM MitoTracker Green (ThermoFisher) was added. Cultures were grown for a further 30 minutes. Cells were collected by centrifugation and suspended in KS buffer with 3% formaldehyde. Cells were washed twice with PBS, collected by centrifugation and suspended in PBS with 1% BSA. Cells were strained through 40 μm mesh immediately prior to cytometric analysis. MitoTracker Green fluorescence was measured with a BD LSRFortessa flow cytometer and the data was analyzed with FlowJo.
Mitotracker green
Manufactured by BD
MitoTracker Green is a fluorescent dye used for staining mitochondria in live cells. It selectively accumulates in active mitochondria, allowing for the visualization and analysis of mitochondrial structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker green, by Thermo Fisher Scientific (3 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by BD (1 mentions)
Lsrii green 532 flow cytometer, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Cellquest pro, by BD (1 mentions)
Mitosox, by BD (1 mentions)
3 protocols using mitotracker green
1
Mitochondrial Staining in S. cerevisiae
2
Fluorescent Mitochondrial Membrane Potential Assay
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Cells were treated with HG, T2E and RAD as described above. After 5 days post-radiation, cells were trypsinized and washed with PBS once. Cells were treated with 100 nM of both Mitotracker Red CMXRos (Invitrogen, Molecular probes, cat #M7512) and Mitotracker Green (Invitrogen, Molecular probes, cat #M7514) in serum free media for 20 min at 37 °C in 5% CO2 in the dark, followed by washing with HBSS buffer three times. Cells were subjected to flow cytometry using a LSRII Green 532 Flow Cytometer (BD Biosciences, San Jose, CA) using the Y/G 605/15 laser for Mitotracker Red CMXRos (MTR) and blue 530/30 laser for Mitotracker Green (MTG). We have used carbonyl cyanide m-chlorophenylhydrazone (CCCP) as a positive control for altering mitochondrial membrane potential. We treated P3158 cells with 10 μM CCCP for 1 h before staining the cells with Mitotracker Green and red.
3
Mitochondrial Membrane Potential Assay
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AML12 cells exposed to Opti-ELF-WMF for the indicated time periods were washed with PBS. MitoSOX (M36008, Thermo Scientific) and MitoTracker Green (M7514, Thermo Scientific) were dissolved in Hank’s balanced salt solution (HBSS, Gibco) at 5 µM and 50 nM, respectively. TMRM was dissolved in the medium at 200 nM. Each dye was added to the cells and incubated for 30 min at 37 °C in a humidified incubator. The cells were then washed with PBS, trypsinized, resuspended in PBS, and harvested. Signal intensities of MitoSOX, MitoTracker Green, and TMRM were quantified by FACSCalibur Flow Cytometer (BD Biosciences) according to the manufacturer’s protocols. Data were analyzed with CellQuest Pro (BD Biosciences).
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