The fluorescent probe MitoTracker Green FM (Molecular Probes, Eugene, OR) was used to determine the mitochondrial mass of adipocytes. In brief, the treated adipocytes were incubated with 0.1 μmol/l MitoTracker Green FM in KRH buffer for 30 min at 37°C, then were centrifuged, resuspended and fluorescence was analyzed by flow cytometry (FACS Calibur, Becton Dickinson, Mountain View, CA).
Mitotracker green fm
Manufactured by BD
Sourced in United States
MitoTracker Green FM is a fluorescent dye that selectively stains mitochondria in live cells. It is a membrane-permeant dye that accumulates in active mitochondria. The dye exhibits green fluorescence upon binding to mitochondrial lipids.
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Lab products found in correlation
Mitotracker green fm, by Thermo Fisher Scientific (5 mentions)
Facscalibur, by BD (1 mentions)
Dcf da, by BD (1 mentions)
Facscan, by BD (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Keyence (1 mentions)
Facscanto 2 flow cytometry system, by BD (1 mentions)
Facscanto facs canto 2, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Anti mouse cd8 clone 53 6, by BD (1 mentions)
Anti human cd3 ucht1, by BioLegend (1 mentions)
6 protocols using mitotracker green fm
1
Mitochondrial Mass Quantification in Adipocytes
2
Oxidative Stress Assays in Cells
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Cells were stained with 20 μM DCFDA (Sigma) or 400nM MitoTracker Green FM (invitrogen) in minimum essential medium without serum for 30 min at 24 h after the last FR at indicated days. DCFDA-, MitoSOX-red- or MitoTracker Green FM-stained cells were quantified with a FACScan (Becton Dickinson, USA). Cells were placed on glass slides and cultured overnight. Cells on coverslips were stained with MitoSOX-red or MitoTracker Green FM according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Images were acquired using a CCD camera attached to a fluorescence microscope (Keyence, Osaka, Japan).
3
Mitochondrial Function in C2C12 Myoblasts
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Following 72 h doxycycline induction, C2C12 myoblasts were seeded onto six-well plates at the density of 15,000 cells/cm2. The next day, cells were treated with 2 μg/mL recombinant Mstn protein or an equal volume of dialysis buffer (control) and incubated at 37°C, 5% CO2. After 24 h, myoblasts were stained with 150 nM MitoTracker Green FM (Thermo Fisher Scientific, Waltham, MA) for 20 min at 37°C. Cells were washed twice with PBS and harvested in conical tubes by centrifuging at 300 g for 1 min. Cell pellets were resuspended in PBS, and FACS analysis was performed to detect MitoTracker Green FM fluorescence intensity using the FACSCanto II flow cytometry system (BD Biosciences, Franklin Lakes, NJ). Fluorescent intensity of 10,000 events from 3 replicate wells per experimental group was detected using the FITC channel and is represented as mean fluorescent intensity. Two technical replicates were performed.
4
Quantifying Mitochondrial Content via MitoTracker
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The mitochondrial content was measured using the MitoTracker™ Green FM (Invitrogen™, M7514). The selection of MitoTracker™ Green FM is based on its ability to be essentially nonfluorescent in aqueous solutions and exhibit fluorescence once it accumulates in the lipid environment of mitochondria. Therefore, the false-positive results are negligible, enabling a correct count of mitochondria via bright green, fluorescein-like fluorescence. Briefly, cells were cultured and treated as indicated, trypsinized, spun down at 400 rpm, and resuspended in buffer made from PBS and FBS (3:1) with MitoTracker™ Green FM at a concentration between 100–400 nM. After incubating between cells with dye for 15–30 min at 37 °C, mitochondria were analyzed using the BD FACSCanto™ (BD FACS Canto II) flow cytometry cell analyzer (BD Biosciences, San Jose, CAL, USA). The FlowJo software was used for the analysis of flow cytometry data (Tree Star, Inc. OR, USA).
5
Mitochondrial Mass Quantification by Flow Cytometry
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Mitochondrial mass was measured by MitoTracker Green FM (Molecular Probes) staining (50 , 52 (link)). Cells were trypsinized and resuspended in PBS with 200 nM MitoTracker Green FM (30 min at 37°C) in the dark and were analyzed by flow cytometry (BD, FACSAria III).
6
Mitochondrial Imaging and Analysis
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To label mitochondria, 100-500 nM MitoTracker Red CMXRos for HeLa cells was used. Images of live cells in L-15 on poly-L-lysine coated ΔTs were acquired on an inverted microscope. Images of fixed OptoMito-On expressing HeLa cells were taken on a confocal microscope. During CD8+ T cell activation, mitochondrial mass was determined with 200 nM MitoTracker Green FM and mitochondrial membrane potential with 20 nM TMRM. Background TMRM staining was subtracted by adding 10 μM FCCP while staining with TMRM. Cells were stained with MitoTracker Green FM or TMRM for 15 minutes at 37°C then stained with anti-mouse CD8 (Clone 53-6.7, BD Biosciences) or anti-human CD3 (UCHT1, Biolegend) followed by flow cytometry.
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