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).
Facscanto facs canto 2
Manufactured by BD
Sourced in United States
The BD FACSCanto™ (BD FACS Canto II) is a flow cytometer designed for the analysis of cells and other particles. It is capable of measuring multiple parameters of individual cells or particles passing through a laser beam. The instrument can detect and quantify various cellular characteristics, such as size, granularity, and fluorescence, enabling researchers to obtain detailed information about cell populations.
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2 protocols using facscanto facs canto 2
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Quantifying Mitochondrial Content via MitoTracker
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Mitochondrial Content Analysis by Flow Cytometry
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Flow cytometry was performed at the University of Alberta’s flow cytometry core facility. The content or density of mitochondrial in cells was measured by the MitoTracker™ Green FM (Invitrogen™, M7514) based on the manufacturer’s instructions using a flow cytometer (FACS Canto II, BD Bioscience, CA, USA). Briefly, cells were cultured and treated as indicated above. After treatment, cells were trypsinized, collected, and resuspended in buffer made from PBS and FBS (3:1) with MitoTracker™ Green FM at 400 nM concentration. After incubation with dye for 45 min at 37 °C, mitochondrial content was 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).
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