C2C12 myoblasts were seeded in 6-well plates and treated with UCF101 or DMSO after differentiation was initiated. The cells were harvested at D1, 3, and 5 and stained according to the manufacturer’s instructions. Apoptosis was evaluated using an Annexin V-FITC PI double staining kit (BD Biosciences, San Diego, CA, USA). The number of mitochondria was measured using a MitoTracker Green probe (Beyotime, Shanghai, China). ROS levels were measured using a Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China). Samples were analyzed by flow cytometry (Guava easyCyte, Austin, TX, USA).
Mitotracker green probe
Manufactured by Beyotime
Sourced in China
MitoTracker Green probe is a fluorescent stain that selectively labels mitochondria in live cells. It is a lipophilic dye that accumulates in active mitochondria based on their membrane potential. The probe binds to mitochondrial proteins, allowing for the visualization and localization of mitochondria within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc pi double staining kit, by BD (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
C1027, by Beyotime (1 mentions)
Jc 1 fluorescent probe, by Beyotime (1 mentions)
Laser confocal microscopy, by Olympus (1 mentions)
Mitochondria isolation kit, by Beyotime (1 mentions)
Dragonfly 200, by Oxford Instruments (1 mentions)
Bodipy 558 568, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
8 protocols using mitotracker green probe
1
Apoptosis and Mitochondrial Dynamics Evaluation in C2C12 Myoblasts
2
Visualizing Mitochondrial Dynamics with Cu-Cy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
When cells reached approximately 80% confluence, they were pretreated with Cu-Cy(100 mg/L) in RPMI-1640 medium in a humidified atmosphere of 5% CO2 at 37 °C for 4 hours. Next, the cells incubated for 30 minutes with 150 nM MitoTracker Green probe (China, Beyotime). Cells were washed with PBS and then immediately observed under CLSM (Germany, Leica). The excitation wavelength for Cu-Cy was 360 nm. The fluorescence excitation and emission wavelengths of the MitoTracker probe are 490 nm and 526 nm, respectively.
3
Quantifying Mitochondrial Content via qRT-PCR and Fluorescence Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantify mitochondrial content, we tested mtDNA content and used Mito-Tracker Green probe (Beyotime, C1048). mtDNA was quantified via real-time fluorescent quantitative PCR (qRT-PCR) by measuring the ratio of Cox2 gene to an intron of the β-globin gene. The Mito-Tracker Green probe was added to the cell culture medium and incubated at 37 °C for 30 min. Mito-tracker Green staining working solution was removed, and fresh cell culture solution preincubated at 37 °C was added. The mitochondrial staining was then observed with a fluorescence microscope.
4
Mitochondrial Imaging in Living Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MitoTracker Green is a green fluorescent probe that can be used for mitochondria‐specific fluorescence in living cells. The transfected cells were incubated in a pre‐warmed 37°C staining solution containing 200 nM MitoTracker Green probe (C1048, Beyotime) for 30 min. Following incubation, the samples were washed three times to remove the residual probe. Next, cells were stained with 1× Hoechst staining solution for live cells (C1027, Beyotime) for 10 min, which binds to the cell nucleus, and rinsed with medium droplets three times. The cells were observed to evaluate the mitochondrial distribution using a confocal microscope.
5
Mitochondrial Dynamics and Bioenergetics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Both 10 μg/ml JC-1 fluorescent probe (Beyotime, China) and 20 nm MitoTracker Green probe (Beyotime, China) were performed in present study to accurately determine the mitochondrial membrane potential (ΔΨm) and mitochondrial morphology. In addition, 10 μM 2,7-dichlorofuorescin diacetate (DCFH-DA) (Nanjing Jianchang, China) fluorescent probe were used to assess intracellular ROS levels by fluorescence microscopy (Thermo Fisher Scientific, USA). While mitochondrial ROS (mito-ROS) levels were loaded with 5 μM MitoSOX Red (ABclonal Technology, China) and visualized by Laser confocal microscopy (Olympus Optical, Japan) and fluorescence microplate reader [27 (link)]. For ATP detection, the cellular ATP content and NA+-K+ -ATPase activity were assessed by ATP assay kit (Product No.A095-1-1, Nanjing Jianchang, China). The measurement of mitochondrial ATP (mito-ATP), mitochondria were isolated using a mitochondria isolation kit (Beyotime, China) according to the instruction manual and then detected using an ATP assay kit (Product No.A095-1-1, Nanjing Jianchang, China) [28 (link)].
6
Mitochondrial Staining and Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondria went through staining using MitoTracker Green probes (Beyotime Biotechnology) per instructions. The study utilized a high-speed confocal platform (Dragonfly 200, Andor, UK) for capturing fluorescent images. The fluorescence intensities were corrected for the protein concentration.
7
Cell Labeling and Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The A549 and H1299 cells were prepared and blocked with rat IgG (10 μg/mL; Sigma) for at least 20 min on ice. After that, they were washed with staining buffer (PBS containing 2% FBS). Then, the cells were labeled with the indicated antibodies (1:100) for 30 min at 4 °C. Dead cells were excluded using a Fixable Viability Dye Efluor 780 (1:1000; Cat No. 65–0865-14, eBioscience). Bodipy558/568 (Cat No. D3835) was obtained from Thermo Fisher Scientific. Mito-Tracker Green probes (Cat No. C1048) were from Beyotime (Shanghai, China). Flow cytometry (FCM) was performed on BD FACS Canto II platforms, and the results were analyzed with FlowJo software version 10.0.7 (TreeStar). Sorting was performed on a BD FACSAria II instrument (BD Biosciences).
8
Mitochondrial Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were subjected to mitochondrial labeling using MitoTracker Green probes (Beyotime, Shanghai, China, C1048) based on the manufacturer's guidelines. Afterward, the live cells that had been labeled were analyzed using confocal imaging to quantify the mitochondria present. Fluorescence intensity was analyzed by Image J software and corrected by protein concentration.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!