The mitochondria in the primary cardiomyocytes were labeled with MitoTracker Red CMXRos probe (100 mmol/L, Invitrogen, Carlsbad, USA) and imaged using a confocal laser-scanning microscope (Nikon A1R MP + Confocal Microscope). The number and volume of the mitochondria observed were analyzed and quantified as previously described [17 ]. After MitoTracker Red staining, flow cytometry was performed to analyze mitochondrial mass. MitoSOX staining (Invitrogen, Carlsbad, USA) was performed to evaluate mitochondrial ROS levels in cardiomyocytes. Intracellular ATP content was detected using an ATP bioluminescent assay kit (Biovision, California, USA) according to the manufacturer's protocol.
Atp bioluminescent assay kit
Manufactured by Abcam
Sourced in United States
The ATP bioluminescent assay kit is a laboratory tool used to quantify the presence and concentration of adenosine triphosphate (ATP) in a sample. It utilizes the bioluminescent reaction between ATP and the enzyme luciferase to generate light, which is then measured to determine the ATP level.
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2 protocols using atp bioluminescent assay kit
1
Quantifying Mitochondrial Dynamics in Cardiomyocytes
2
Mitochondrial Function Assays in HepG2 Cells
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For all determinations, HepG2 cells were seeded in 96 well plates and treated with E2 as previously described.
Values were normalized per number of viable cells determined by crystal violet nuclear staining assay (Nagamine et al. 2009) (link). Mitochondrial mass was assessed using the fluorescent probe N-nonyl acridine orange (NAO) as it specifically stains mitochondrial phospholipid cardiolipin (Petit et al. 1992 (link)). Briefly, 232:2 0.5 μM NAO were added to HepG2 cells for 30 min at 37°C in darkness and fluorescence measurement was performed in a FLx800 microplate fluorescence reader (Bio-Tek) with excitation at 485 nm and emission at 528 nm. Mitochondrial membrane potential (MMP) was tested using tetramethylrhodamine methylester (TMRM) as a lipophilic cationic dye that accumulates within mitochondria according to the membrane potential (Scaduto & Grotyohann 1999) (link). Cells were dyed with 0.5 μM of TMRM for 15 min at 37°C in darkness and fluorescence intensity was measured with excitation at 552 nm and emission at 576 nm. Finally, ATP was measured with an ATP bioluminescent assay kit (BioVision) following the manufacturer's instructions.
Values were normalized per number of viable cells determined by crystal violet nuclear staining assay (Nagamine et al. 2009) (link). Mitochondrial mass was assessed using the fluorescent probe N-nonyl acridine orange (NAO) as it specifically stains mitochondrial phospholipid cardiolipin (Petit et al. 1992 (link)). Briefly, 232:2 0.5 μM NAO were added to HepG2 cells for 30 min at 37°C in darkness and fluorescence measurement was performed in a FLx800 microplate fluorescence reader (Bio-Tek) with excitation at 485 nm and emission at 528 nm. Mitochondrial membrane potential (MMP) was tested using tetramethylrhodamine methylester (TMRM) as a lipophilic cationic dye that accumulates within mitochondria according to the membrane potential (Scaduto & Grotyohann 1999) (link). Cells were dyed with 0.5 μM of TMRM for 15 min at 37°C in darkness and fluorescence intensity was measured with excitation at 552 nm and emission at 576 nm. Finally, ATP was measured with an ATP bioluminescent assay kit (BioVision) following the manufacturer's instructions.
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