Cells were infected with S. typhimurium (10:1), AIEC strain LF82 (10:1; generous gift from Dr Emiko Mizoguchi) or S. aureus (1:1) for a total of 2 h, with gentamicin added 1 h after bacterial infection35 (link). In some cases, cells were pretreated for 1 h with 50 nM Mito-Tempo (Santa Cruz) or 20 nM N-acetylcysteine (Sigma).
Mito tempo
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mito-TEMPO is a mitochondria-targeted antioxidant that acts as a superoxide dismutase (SOD) mimetic. It is designed to scavenge superoxide radicals within the mitochondria, providing protection against oxidative stress.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Mitosox red, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Mitotracker green, by Thermo Fisher Scientific (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
Chloroquine, by Merck Group (2 mentions)
Apocynin, by Santa Cruz Biotechnology (2 mentions)
Sb203580, by Santa Cruz Biotechnology (2 mentions)
Sp600125, by Fujifilm (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Z devd fmk, by Merck Group (2 mentions)
D mannitol, by Merck Group (2 mentions)
Cremophore el, by Merck Group (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Gp91ds tat, by AnaSpec (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Tom20, by Santa Cruz Biotechnology (2 mentions)
Mdivi 1, by Merck Group (2 mentions)
Z vad fmk, by Peptide Institute (2 mentions)
46 protocols using mito tempo
1
Infection and Pretreatment Assay
2
Antiviral Autophagy Pathway Analysis
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HEK293 cells, HeLa cells, U2OS cells and Vero cells were cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT, USA) and antibiotics (100 U ml−1 penicillin and 100 μg ml−1 streptomycin, Invitrogen). Antibodies used in this study for western blot or immunofluorescence assays were as follows: mouse anti-Flag (Sigma, St Louis, MO, USA), rabbit anti-LC3 (Novus, Littleton, CO, USA and MBL, Woburn, MA, USA), mouse anti-TIM23 (BD, Waltham, MA, USA), mouse anti-GAPDH and rabbit anti-Caspase-3 (Sungene Biotech, Tianjin, China), rabbit anti-Myc and rabbit anti-HA (MBL), rabbit anti-ATG5 (Epitomics, Cambridge, MA, USA), and rabbit anti-TOM20, mouse anti-Calnexin, mouse anti-TRAF2 and rabbit anti-TRAF6 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Cleaved Caspase-3 (Cell Signalling Technology, Danvers, MA, USA); the antibody against MAVS was produced by our lab [14 (link)]. Mito-TEMPO was purchased from Santa Cruz Biotechnology or Enzo Life Science, Farmingdale, NY, USA. Pyrrolidinedithiocarbamic acid ammonium salt (PDTC), bafilomycin A1 and chloroquine were purchased from Sigma. VSV was propagated in Vero cells.
3
Mitochondrial Superoxide and Apoptosis
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MLE-12 cells were pretreated with 10 µM MitoTEMPO (Santacruz, TX, United States) or vehicle control for 2 h and subjected to NO or HO for 24 h and then assessed for mitochondrial superoxide and apoptosis.
4
Diverse Pharmacological Inhibitors Assay
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Doxorubicin (Dox) was purchased from Sigma. Cisplatin, anisomycin, propyl gallate (PG), SP600125, N-acetylcysteine (NAC) and U0126 were purchased from Wako. SB203580, Etoposide, Apocynin (Apo) and mitoTEMPO (MT) were purchased from Santa Cruz. NSC87877 and wortmannin were purchased from Cayman.
5
Metabolic Regulation in Cancer Cells
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Unless specified, all reagents were obtained from Sigma and all the antibodies were from Santa Cruz Biotechnology, except for anti-HIF-1α (Becton Dickinson), anti-PKM2 and anti-Src-Tyr416 (Cell Signaling Technology) and anti phospho-Tyr (clone 4G10) (Millipore). HRP-conjugated streptavidin was from Pierce. HIF-1-siRNA and DEC1-siRNA were from Santa Cruz Biotechnology. N-(biotinoyl)-N-(iodoacetyl)ethylenediamine (BIAM) and 2′-7′-dichlorofluoresceindiacetate (H2-DCF-DA) were from Molecular Probe. [U-14C] lactate and [U-14C] glucose were from Perkin Elmer. All the kits used to perform miRNA extraction and quantitative reverse transcriptase PCR were from Qiagen. Lipofectamine 2000 was from Invitrogen. Metformin was obtained by Sigma. The chemical synthesis of the PKM2 activator DASA-58 was kindly performed by Dr. Richichi of the Department of Chemistry (University of Florence). The mitochondrial antioxidant MitoTEMPO was from Santa Cruz Biotechnology. The MCT1 inhibitor, AR-C155858, was from Tocris Bioscience.
6
Isolating PBMC from CKD-HD patients
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Freshly isolated PBMC from 3 randomly selected CKD-HD were cultured in RPMI 1640 medium (Sigma) supplemented with 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml) and stimulated with LPS (10 ng/ml) (Sigma) for 4 h followed by ATP (Sigma) 1 mM for 15 min. MitoTEMPO (100 μM) (Santa Cruz) was added to the medium 1 h before LPS priming.
7
Assay Reagents for Oxidative Stress
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Water-soluble progesterone (PG), anesthetic MS-222 and apocynin were obtained from Sigma (St. Louis, MO, USA). Collagenase (280 U/mg) and catalase (11,000 U/mg) were purchased from Wako (Osaka, Japan), hCG was from Teikoku Zoki (Tokyo, Japan). Fluorogenic caspase-3 substrate IV was purchased from Calbiochem (La Jolla, CA, USA). Hydrogen peroxide colorimetric/fluorometric assay kit was from BioVision (Milpitas, CA, USA). Polyclonal anti-MAPK and anti-pMAPK antibodies were from Cell Signaling (Beverly, MA, USA), biotinylated anti-rabbit IgG was from Vector Laboratories (Burlingame, CA, USA). The Streptavidin Biotin Complex Peroxidase Kit, protein assay CBB solution, SOD (5000 U/mg) and BHA were from Nacalai Tesque (Kyoto, Japan). 2′,7′-dichlorofluorescein diacetate (DCFDA) Cellular ROS Detection Assay Kit was obtained from Abcam (Cambridge, UK), EUK 134 and MITO-TEMPO were ordered from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA). Other chemicals were obtained from Wako and Nacalai Tesque.
8
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was measured using the potential-sensitive dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide). Briefly, RAW264.7 cells (1 × 104) were plated in a 24-well plate and allowed to attach overnight. shNC or shSOD2 lentiviruses were added into the wells and cultured for 48 h under normoxia, and the cells were then exposed to IH for 48 h with or without mito-TEMPO (Santa Cruz, sc-221945) treatment (100 nM, 2 h before IH exposure). The cells were incubated with medium containing JC-1 (10 μg/mL) for 20 min at 37 °C. Then, the cells were washed with PBS and observed by confocal microscopy or measured by the quantitation of fluorescence intensity through flow cytometry.
9
Senescence Assay for Vascular SMCs
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VSMC senescence was assessed based on SA-β-gal staining according to the manufacturer’s protocol (Beyotime, C0602). Briefly, control VSMCs and MFS VSMCs were plated in a 6-well culture plate. Some of the control cells were treated for 48 h with 50 ng/ml TGF-β1 (PeproTech, 100-21) combined with 100 μM Mito-tempo (Santa Cruz, SC-221945) or with 1 μM SC-54, a NF-κB inhibitor (Sigma-Aldrich, SML0557). The cells were then washed with PBS, fixed for 30 mins, and stained with SA-β-gal staining solution over night at 37°C (without CO2). After washing three times with PBS, the cells were randomly photographed.
10
Chemical Modulation of Cellular Processes
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E-64D, pepstatin A, Chloroquine, rapamycin, 3-MA, ascorbic acid, or β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA) were applied to cell culture media to treat cells at the indicated concentrations and for the indicated periods of time. Hydrogen peroxide was purchased from Wako (Osaka, Japan). LysoTracker, MitoTracker, and Mito-TEMPO were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
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