MCF-7 and MCF-10A cells were seeded at a density of 20,000 cells/well into 48-well plates and allowed to grow for 24 h. The cells were then treated with single and combination treatments of CA, CGA, and Arc, or fresh medium for the untreated group, for 6 h and 24 h. After 6 h or 24 h, the cells were incubated with MitoTracker® Green FM (Sigma–Aldrich, Saint Louis, MO, USA) to a final concentration of 150 μM of MitoTracker® Green FM for 30 min and Hoechst 33342 to a final concentration of 1 μg/mL for 15 min at 37 °C by direct addition to cell medium and subsequent mixing. After incubation, the cells were washed once with warm PBS and FluoroBriteTM DMEM medium (Sigma–Aldrich, Saint Louis, MO, USA) was added for cell imaging. The cells were imaged using the EVOS™ M7000 Imaging System (Invitrogen, Waltham, MA, USA). The cells were visualized with a 40× objective and DAPI (Ex: 357/44; Em: 447/60) and GFP (Ex: 482/25; Em: 524/24) filter cubes. All settings were kept equal between samples of the same cell lines and between biological replicates. The images were processed and analyzed using FIJI [97 (link)]. All brightness/contrast adjustments were kept equal between all samples of the same cell lines. The MCF-7 cell area was determined by manually drawing around individual cells in phase–contrast images and measuring the area in FIJI.
Mitotracker green fm
Manufactured by Merck Group
Sourced in United States
MitraTracker Green FM is a fluorescent dye that specifically stains the mitochondria in live cells. It is a cell-permeant, non-toxic dye that accumulates in active mitochondria. The dye exhibits green fluorescence upon binding to mitochondrial membranes.
Automatically generated - may contain errors
Lab products found in correlation
Bafilomycin a1 baf, by Merck Group (2 mentions)
Axiocam mrc scope a1, by Zeiss (2 mentions)
Tcs sp5, by Leica (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Mitotracker green fm, by Beyotime (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
Evos m7000 imaging system, by Thermo Fisher Scientific (1 mentions)
Cytoflex platform, by Beckman Coulter (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Thioltracker violet, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
9 protocols using mitotracker green fm
1
Mitochondrial Dynamics in Breast Cancer Cells
2
Mitochondrial Network Analysis in M1/M2-Polarized BMDMs
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BMDM were seeded on confocal microscopy sectored dishes (5×104 cells per sector) and then M1/M2-polarized as described above. Cells were washed following stimulation and stained with 100 nM MitoTracker Green FM, and 2 μg/mL Hoechst 33342 (Sigma Aldrich, USA) for 30 min. Microscopy was performed using Leica TCS SP5 (Leica, Germany) confocal microscope. For analysis of mitochondrial network morphology, Image-J compatible MiNA tool was used (38 (link)).
3
Quantifying Mitophagy Flux via Flow Cytometry
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Mitophagy flux was evaluated as described previously [28 (link)]. Briefly, cells were stained with MitoTracker Green FM (500 nM, C1048, Beyotime, Shanghai, China), and analyzed by using CytoFlex platform (Beckman Co., Ltd, South Kraemer Boulevard Brea, CA, USA). Mitophagy flux was defined as the ratio of MitoTracker Green FM fluorescence in the presence of mitophagy and lysosomal inhibitor (bafilomycin A1, Baf, 10 nM, Sigma-Aldrich, St. Louis, MO, USA) to that in the absence of inhibitor, normalized to the corresponding value in control cells.
4
Mitochondrial Activity Assessment in Yeast
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The mitochondrial activity was monitored using the Rhodamine 123 (Sigma-Aldrich) and Mitotracker Green FM (Sigma-Aldrich) fluorescent dyes. Yeast cells were stained according to the manufacturer’s instructions. Initially, yeast cells were harvested by centrifugation of 8000 × g for 10 min at 4°C, diluted in PBS 1X to 106 cells/mL and stained during 45 min at 37°C with Mitotracker (400nM). Then, the cells were washed with PBS 1 X and labeled with Rhodamine (2.4 μM) for 45 min at 37°C. Afterward, the cells were washed three times with PBS 1X and analyzed by fluorescence microscopy (Zeiss Axiocam MRc – Scope A1) at 450–490 nm for Mitotracker (FS09) and 515–575 nm filter (FS15) for Rhodamine. All experiments were performed in triplicate and the minimum of 50 cells for each microscope slides were assessed to measure of the fluorescence intensity (in pixels). Student’s t-test and p ≤ 0.05 were considered statistically significant as described by Chaves et al. (2019) (link).
5
Verifying Nanodiamond Internalization in Cells
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We take particular caution in verifying
the nanodiamonds are internalized inside the cells, to avoid measuring
nanodiamonds attached to the surface of the cell. To achieve this,
a series of 3D confocal scanning images on a Leica SP5 are taken to
establish that the nanodiamonds are situated among the mitochondrial
network which has been dyed with 100 nM MitoTracker Green FM (Sigma-Aldrich,
UK) (Supporting Information Section 9 ).
The observation that there are mitochondria above, below, and surrounding
the nanodiamond observed confirms that the nanodiamond is internalized.
the nanodiamonds are internalized inside the cells, to avoid measuring
nanodiamonds attached to the surface of the cell. To achieve this,
a series of 3D confocal scanning images on a Leica SP5 are taken to
establish that the nanodiamonds are situated among the mitochondrial
network which has been dyed with 100 nM MitoTracker Green FM (Sigma-Aldrich,
UK) (
The observation that there are mitochondria above, below, and surrounding
the nanodiamond observed confirms that the nanodiamond is internalized.
6
Mitophagy Flux Evaluation Protocol
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Mitophagy flux was evaluated as a previous study described. In brief, MitoTracker Green FM (500 nM) was used to stain cells. A CytoFlex platform (Beckman, South Kraemer Boulevard, Brea, CA, USA) was used for analysis. Mitophagy flux was defined as the ratio of MitoTracker Green FM fluorescence in the presence of mitophagy and lysosomal inhibitor (bafilomycin A1, Baf, 10 nM, Sigma-Aldrich, St. Louis, MO, USA) to that in the presence of only mitophagy.
7
Mitochondrial activity of mebendazole-exposed cells
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The mitochondrial activity of cells exposed or not to mebendazole for 12 h were verified through fluorescence microscopy using MitoTracker Green FM (Sigma-Aldrich) dye. Samples were collected and incubated for 45 m at room temperature in the dark with MitoTracker Green FM (400 µL) and then washed three times with PBS. Fluorescence analyzes were performed in triplicate and visualized using a fluorescence microscope (Zeiss Axiocam MRc-Scope A1) with a wavelength of 450–490 nm. Pixel quantification was performed with AxioVision software (Carl Zeiss), where all fluorescent and well-delimited cells were analyzed. The static difference was evaluated by the Student’s t test, where the difference was considered significant when p ≤ 0.05.
8
Quantifying Muscle Stem Cell Metabolism
9
Immunofluorescence Assay for Macrophages
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CPT (C5624), LPS, MitoTracker Green FM (M7514), and bafilomycin A1 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The fluorescein isothiocyanate-conjugated secondary antibodies were obtained from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA) and murine recombinant proteins macrophage colony stimulating factor (M-CSF), IFN-γ, and IL-4 were purchased from Peprotech (NJ, USA).
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