Mitochondrial damage was evaluated by a flow cytometry-based assay as previously described24 (link). In brief, macrophages were first primed with LPS, followed by ATP or Nigericin treatment for 30 min, after which the cells were stained with 100 nM MitoTracker Deep Red and 100 nM MitoTracker Green (Cell Signaling Technology) for 15–30 min. The cells were then washed and analyzed on a BD FACSymphony A3 Cell Analyzer. Data analysis was performed using FlowJo version 10 software (Tree Star). Gating strategy for flow cytometric analysis is shown in Supplementary Fig. 4 .
Mitotracker deep red
Manufactured by Cell Signaling Technology
MitoTracker Deep Red is a fluorescent stain used to detect mitochondria in live cells. It passively diffuses across the plasma membrane and accumulates in active mitochondria. The stain emits red fluorescence upon binding to mitochondrial membranes.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Mitotracker green, by Cell Signaling Technology (1 mentions)
C0602, by Beyotime (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Mouse anti flag m2 antibody, by Merck Group (1 mentions)
Anti flag m2 agarose, by Merck Group (1 mentions)
3t3 l1 atcc cl 173, by American Type Culture Collection (1 mentions)
Rabbit anti flag m2 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti calnexin, by Stressgen (1 mentions)
Anti myc tag antibody, by Merck Group (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Er tracker red, by Thermo Fisher Scientific (1 mentions)
8 protocols using mitotracker deep red
1
Assessing Mitochondrial Damage in Macrophages
2
Senescence and Mitochondrial Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TKPTS cells cultured on plates were performed SA‐β‐gal,MitoTracker, and TMRE staining according to the guides of the manufacturer. SA‐β‐gal staining was used to test the β‐galactosidase activity (C0602, Beyotime Biotechnology, Shanghai, China) for the detection of senescence. MitoTracker deep red (9082P, Cell Signaling Technology) and Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE, T669, Thermo Fisher Scientific) staining were adopted to measure mitochondria and mitochondrial membrane potential. TKPTS cells were incubated in MitoTracker (100 nM) or TMRE (200 nM) at 37°C against the light for 30 min before performing the fluorescence activation.
3
Mitochondrial Imaging of SIRT1/PGC-1α Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HT22 cells were seeded in 6-well plates. After treatment with SIRT1 or PGC-1α activator/inhibitor, the cells were incubated with MitoTracker Deep Red (500 nM, Cat# 8778S, Cell Signaling Technology) for 30 min at 37 °C. Images were captured by confocal microscopy (LSM 800, Carl Zeiss).
4
Galectin-12 Antibody Generation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polyclonal galectin-12 antibodies were generated in galectin-12 knockout mice [17 (link)]. Mouse anti-FLAG M2 antibody and Anti-FLAG M2-agarose were purchased from Sigma. They were used for Western blotting and immunoprecipitation of Flag-tagged proteins. For immunofluorescence, rabbit anti-Flag M2 antibody (Cell Signaling Technology) was used. Mouse anti-LAMP1 and anti-Myc tag antibodies were from Millipore and Syd Labs, respectively. MitoTracker Deep Red, rabbit anti-LAMP1 and anti-Myc tag antibodies were purchased from Cell Signaling Technology. Rabbit anti-perilipin-1 was from Affinity Bioreagents. Rabbit anti-calnexin was purchased from Stressgen. The following cell lines were purchased from ATCC: mouse fibroblast cell line 3T3-L1 (ATCC CL-173), human embryonic kidney cell line 293T (ATCC CRL-11268), and the human cervical carcinoma cell line HeLa (ATCC CCL-2). They were cultured following standard conditions in DMEM/10% FBS at 5% CO2, 37°C.
5
Mitochondrial Activity Quantification in BMDM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For FACS analysis, 1×106 BMDM or ER-Hoxb8 cells were used per sample. Mitochondrial activity was determined by staining of cells for 30 minutes with 100 nM MitoTracker Deep Red (Cell Signaling Technologies, Leiden, The Netherlands). Cells were then washed three times with 1x PBS and the mean fluorescence intensity (MFI) was measured in the APC channel.
6
Mitochondrial Membrane Potential and Content
7
Organelle Colocalization via Fluorescent Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Colocalization experiments were performed with the following organelle markers post Laurdan staining for ER, Lysosomes and Mitochondria, respectively: ER-Tracker Red™ (1 µM) (Thermo Fisher), LysoTracker™ Deep Red (1 µM) (Thermo Fisher), MitoTracker Deep Red (500 nM) (Cell Signaling). To assess Golgi-Laurdan localization, cells were transfected with a SiT-mApple plasmid using Lipofectamine 3000 (Thermo Fisher). Briefly, 12 hours post seeding, media was aspirated and washed with HBSS and incubated in OptiMEM (Thermo Fisher) for 15 minutes. Lipofectamine 3000 plasmid mixtures were added, and cells were incubated for 4 hours. Media was replaced before being stained with the Golgi-Laurdan as described above.
8
Mitochondrial Membrane Potential and Content
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were loaded with either tetramethylrhodamine, ethyl ester (TMRE) (Invitrogen: #T669) to measure mitochondrial membrane potential or MitoTracker Deep Red (Cell Signaling: #8778S) to label mitochondria following the manufacturer’s protocols. After loading, the cells were collected by trypsinization, resuspended in 2% FBS in PBS, filtered and subjected to flow cytometry analysis. Flow cytometry data were recorded on an LSR II (BD Bioscience) for 10,000 events. To measure mitochondrial membrane potential, fluorescence intensity data was collected using ex. 561 nm, em. 582±15 nm. To control for TMRE loading, FCCP was added to collapse the mitochondrial membrane potential after collecting the baseline recording. The FCCP recording was subtracted from the baseline recording. To measure mitochondrial content in intact cells, the MitoTracker Deep Red fluorescence intensity data were collected using ex. 640 nm, em. 660±10 nm.
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!