After 48 h of intervention, the changes in ROS levels of MC3TE-E1 cells following the intervention were evaluated using 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma, St. Louis, MO) staining and MitoSOX™ Red mitochondrial superoxide indicator (MitoSOX™; Sigma, St. Louis, MO) staining.
To confirm the changes in mitochondrial membrane potential, we use co-stained with tetramethylrhodamine methyl ester (TMRM, 100 nM, Life Technologies) and Mitotracker Green (Mitogreen, 100 nM, Life Technologies) to observe and evaluate the state of the mitochondrial oxidative stress in treated cells. After 48 h of intervention, TMRM and Mitogreen staining were performed as previously described [30 (link)]. The results of TMRM (excitation wavelengths = 543 nm) and Mitogreen (excitation wavelengths = 488 nm) were captured using Laser confocal microscope. The quantification of mitochondrial membrane potential was determined using Image J software.
Immunofluorescence staining was used to quantitatively detect and evaluate the expression of SIRT1 and SOD2 in MC3T3-E1 after different interventions to understand the changes of cellular oxidative stress as stated above. The levels of Malondialdehyde (MDA) and total SOD activity in the cells were measured by a malondialdehyde Detection Assay kit (APPLYGEN) and Superoxide Dismutase Activity Assay Kit (Abcam), respectively.
To confirm the changes in mitochondrial membrane potential, we use co-stained with tetramethylrhodamine methyl ester (TMRM, 100 nM, Life Technologies) and Mitotracker Green (Mitogreen, 100 nM, Life Technologies) to observe and evaluate the state of the mitochondrial oxidative stress in treated cells. After 48 h of intervention, TMRM and Mitogreen staining were performed as previously described [30 (link)]. The results of TMRM (excitation wavelengths = 543 nm) and Mitogreen (excitation wavelengths = 488 nm) were captured using Laser confocal microscope. The quantification of mitochondrial membrane potential was determined using Image J software.
Immunofluorescence staining was used to quantitatively detect and evaluate the expression of SIRT1 and SOD2 in MC3T3-E1 after different interventions to understand the changes of cellular oxidative stress as stated above. The levels of Malondialdehyde (MDA) and total SOD activity in the cells were measured by a malondialdehyde Detection Assay kit (APPLYGEN) and Superoxide Dismutase Activity Assay Kit (Abcam), respectively.
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