Mitochondrial membrane potential (Δψm) was analyzed by cationic dye 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimi‐dazolylcarbocyanine iodide (JC‐1, Invitrogen) and MitoTracker Red (CMXRos; Molecular Probes, Carlsbad, CA, USA) staining. Briefly, NAM treated cells were incubated with JC‐1 for 20 min followed by two washes using PBS prior to emission measurements. The emission was measured at 590 and 540 nm after excitation at 480 nm using a multimode microplate reader (Glomax® Explorer; Promega, Madison, WI, USA). For MitoTracker Red analysis, cells were stained with MitoTracker Red (5 μm ) for 30 min at 37 °C and analyzed by flow cytometry and processed using the flowjo™ 10 software (BD Biosciences, San Diego, CA, USA). Fluorescent images of the cells were taken under a fluorescent microscope (Leica, Zeiss, Germany).
Mitotracker red
Manufactured by BD
Sourced in United States
MitoTracker Red is a fluorescent dye that specifically accumulates in active mitochondria. It can be used to label and track mitochondria in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Agarose, by Merck Group (2 mentions)
Ethidium bromide, by Merck Group (2 mentions)
Acridine orange, by Merck Group (2 mentions)
Jc 1 dye, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
Foetal bovine serum (fbs), by HiMedia (1 mentions)
Cell culture dishes and plates, by Corning (1 mentions)
Phosphate buffer saline (pbs), by HiMedia (1 mentions)
Proteinase k, by HiMedia (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Caspase 9 colorimetric kit, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by HiMedia (1 mentions)
Sodium azide, by HiMedia (1 mentions)
Trypsin, by HiMedia (1 mentions)
Dimethyl sulfoxide (dmso), by HiMedia (1 mentions)
Rnase a, by HiMedia (1 mentions)
Fluorescent microscope, by Leica (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
5 protocols using mitotracker red
1
Mitochondrial Membrane Potential Analysis
2
Mitochondrial Response to Dox and PAH6
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A549 cells were seeded in a 24-well plate at a cell density of 60,000 per well and incubated for 24 h before treatment. The cells were then treated with Dox (2 μg/mL), PAH6 (120 μg/mL) or Dox-DNA/PAH6 (11:60) containing 2 μg/mL of Dox for 1 h. The cells were washed twice with warm PBS and then incubated with F12K media containing 300 nM MitoTracker Red (Thermo Fisher, USA) for 20 min under cell growth conditions. All the steps follow the recommendations from the manufacturer’s manual. After the staining step, the staining solution was removed and the cells were washed twice with prewarmed PBS solution and collected. The fluorescence intensity of MitoTracker Red was measured using a BD FACSCalibur flow cytometry (BD Bioscience, San Jose, CA, USA). The mean fluorescence intensity of nontreated cells was counted as 1 to normalize the fluorescence intensity of treated cells.
3
Assessment of Manikya Bhasma's Anticancer Potential
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Manikya Bhasma was obtained from local Baidyanath store in Guwahati city, acridine orange, propidium iodide, ethidium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), agarose, 2,7-dichlorofluorescein diacetate (DCFH-DA), dulbecco’s modified eagle’s medium were purchased from Sigma Aldrich (St. Louis, MO, USA). RNase A, Proteinase K, DMSO, Foetal Bovine Serum (FBS), Penicillin-Streptomycin (100X) antibiotic solution, Phosphate Buffer Saline (PBS), sodium azide, trypan blue, and trypsin were obtained from HiMedia (Mumbai, India). Ethylenediaminetetraacetic acid (EDTA), ethanol, sodium chloride, was purchased from Merck, Germany. Anti-Cyt-c antibodies, Mitotracker Red, and JC-1 dye were obtained from BD-Biosciences (San Jose, USA). The caspase-9 colorimetric kit was obtained from Invitrogen Corporation (Waltham, USA). All the cell culture plates and dishes were purchased from Corning, Lowell, MA, USA. MDAMB-231, DLD-1, HCT-116, MG-63, HeLa cancer cell lines were procured from National Centre for Cell Sciences, Pune, India. All other reagents and chemicals were of analytical grade purity.
4
Immortalized HPMEC Cell Culture and Antibody Analysis
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Immortalized HPMECs were purchased from Applied Biological Materials Incorporation (Richmond, BC, Canada). The cells were cultured in endothelial cell medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin at 37 °C with 5% CO2. Primary antibodies against vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), BAK, HO-1 , NF-κB P65, p-NF-κB P65 and mouse secondary antibody were purchased from Cell Signaling Technology (USA). Bcl2 antibody was obtained from Abcam (USA). β-Actin and Nrf2 antibodies were purchased from Huaan biotechology (China) and Arigo (Taiwan)，respectively. GAPDH was obtained from Beyotime Institute of Biotechnology (China). Purified mouse anti-Cytochrome c and MitoTracker Red were bought from BD Pharmingen (USA) and Invitrogen (Carlsbad, CA, USA), respectively. Rabbit secondary antibody was bought from Beyotime Biotechnology. Pyrrolidinedithiocarbamate ammonium（PDTC）was purchase from Selleck (USA). MG132 and cycloheximide (CHX) were brought from Medchemexpress (USA).
5
Apoptosis and Oxidative Stress Pathways
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N-acetylcysteine (NAC), propidium iodide, ethidium bromide, acridine orange, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), thiobarbituric acid, 1,19,3,39-tetraethoxypropane, guanidine hydrochloride, agarose, DAPI-containing mounting solution, filipin, and chemiluminescence peroxidase kits were purchased from Sigma (St. Louis, USA). Dimethylsulfoxide (DMSO), Triton X-100, Tween-20, hydrogen peroxide, methanol, β-mercaptoethanol, acrylamide, and bis-acrylamide were obtained from Merck (Boston, USA). Anti-PKCα and anti-cyt c antibodies, Mito-Tracker Red, and JC-1 dye were obtained from BD-Biosciences (San Jose, USA). Anti-5′-nucleotidase antibody was purchased from Cell Signaling Technology (Danvers, USA). Caspase-3 assay kit was from BD Pharmingen (San Jose, USA). Caspase-9 colorometric kit was from Invitrogen Corp. (Waltham, USA). Other reagents and chemicals were of analytical grade purity.
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