To test mitochondrial load and membrane potential, mouse embryonic fibroblasts (MEF) were generated from all MNX and wild-type strains. MEF lines ( < passage 3) were harvested and stained individually and in combination with MitoTracker Red CMXRos (Molecular Probes by Invitrogen: M7512) and MitoTracker Green FM (Molecular Probes by Invitrogen: M7514) fluorescent probes followed by analysis by flow cytometry as described previously (20 (link)). Briefly, 100,000 cells from each line were added to cytometer tubes, and then stained with 200 nM of each probe alone or in combination. Cells from each line were also left as unstained controls. Cells were protected from light and the dye incubated for 15 minutes at 37o before placing on ice. Flow cytometry was performed with parameters from Molecular Probes website (MitoRed: Excitation-579 nm and Emission-599 nm, MitoGreen: Excitation-490 nm and Emission-516 nm). Fluorescence intensity at the appropriate emission wavelengths was captured.
Mitored
Manufactured by Thermo Fisher Scientific
MitoRed is a fluorescent dye designed for the selective detection of mitochondria in living cells. It functions by binding to the mitochondrial membrane potential, enabling the visualization and analysis of mitochondrial structure and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Mitogreen, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Lucigenin, by Roche (1 mentions)
Hochest 33342, by Thermo Fisher Scientific (1 mentions)
Cellrox, by Thermo Fisher Scientific (1 mentions)
Human islets, by Lonza (1 mentions)
Complete dmem medium, by Thermo Fisher Scientific (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Palmitate, by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Collagenase p, by Roche (1 mentions)
3 protocols using mitored
1
Evaluating Mitochondrial Dynamics in MEFs
2
Mitochondrial Dynamics in Brown Adipocytes
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In brown preadipocytes, DE was treated for 24 hours and then, tumor necrosis factor-α (TNF-α; 100 ng/mL, 1 hour) treatment. To visualize mitochondrial distribution, brown adipocytes were stained with the mitochondrial marker Mitotracker Red CM-H2XRos (Mito-Red, Molecular Probes) according to the manufacturer’s instructions. Mito-Red was dissolved in a 1:1 mixture of dimethylsulfoxide and saline to a final concentration of 100 nM.
3
Reagents and Cell Lines for Diabetes Research
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DHE, DAPI, CellROX, MitoGreen, MitoRed, MitoSOX, Hochest 33342, and chloromethyl‐2',7'‐dichlorodihydrofluorescein diacetate (CM‐H2DCFDA) were purchased from Molecular Probes (Eugene, OR). Glucose, BSA, trypsin, palmitate, NADPH, and histopaque‐1077 were purchased from Sigma (St. Louis, MO). Lucigenin, and collagenase P were from Roche (Switzerland). GHTT was purified as described previously (Almeida et al, 2011 (link); Kuo et al, 2017 (link)). Recombinant proteins, 6×His‐Pdia4, Gst‐p22phox and Gst‐Ndufs3 were purchased from Enzo (Farmingdale, NY). Gst‐TecSH3, containing a SH3 domain of Tec, was produced as published (Kuo et al, 2017 (link)). The antibodies used in this study were purchased (Appendix Table S3 ). 293T cells (CRL‐3216), Min6 cells (Yagi et al, 1995 (link)), and pancreatic islets were grown in complete DMEM medium (Thermo Fisher, Waltham, MA) containing 10% and 20% fetal bovine serum (FBS), respectively, and 3.3 mM Glucose unless indicated otherwise. Human islets were purchased from Lonza (Switzerland) and handled according to the protocol of the Academia Sinica Institutional Review Board (AS‐IRB01‐14015). Informed consent was obtained from all human subjects and the experiments conformed to the principles of the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
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