Cells were treated with GS at various concentrations (0, 5, 10, and 15 mg/mL) for 48 hrs. For PCr cotreatment, PCr (Laiboten, Harbin, China) was dissolved in DMEM to produce a 2 M stock solution. Then, the cells were pretreated with different concentrations of PCr (0, 2.5, 5, 10, and 20 mM) for 8 hrs, followed by 15 mg/mL GS stimulation for 48 hrs in the presence of PCr. In functional experiments, L-02 cells were treated according to the following groups: (1) Control group: L-02 cells cultured in DMEM; (2) GS group: L-02 cells exposed to 15 mg/mL GS for 48 hrs; (3) GS + PCr group: L-02 cells preincubated with 5 mM PCr for 8 hrs followed by 15 mg/mL GS for 48 hrs in the presence of PCr; (4) PCr group: L-02 cells treated with 5 mM PCr for 56 hrs; (5) Mito-TEMPO group: L-02 cells treated with 50 μM Mito-TEMPO (Sigma, St Louis, MO, USA) for 56 hrs; (6) Mito-TEMPO+GS group: L-02 cells preincubated with 50 μM Mito-TEMPO for 8 hrs followed by 15 mg/mL GS for 48 hrs in the presence of Mito-TEMPO.
Mitotempo
Manufactured by Merck Group
Sourced in United States, Germany, Japan, United Kingdom, Sao Tome and Principe, China, Macao
MitoTEMPO is a mitochondria-targeted superoxide indicator. It is a fluorescent probe designed to measure mitochondrial superoxide levels in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Mitosox red, by Thermo Fisher Scientific (21 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (21 mentions)
Antimycin a, by Merck Group (17 mentions)
Mitosox, by Thermo Fisher Scientific (17 mentions)
N acetyl l cysteine, by Merck Group (16 mentions)
Rotenone, by Merck Group (16 mentions)
N acetylcysteine, by Merck Group (14 mentions)
N acetyl cysteine (nac), by Merck Group (13 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (12 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Mitotracker green, by Thermo Fisher Scientific (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (8 mentions)
N acetyl l cysteine nac, by Merck Group (7 mentions)
Mdivi 1, by Merck Group (7 mentions)
H2dcfda, by Thermo Fisher Scientific (7 mentions)
Bafilomycin a1, by Merck Group (6 mentions)
Palmitate, by Merck Group (6 mentions)
Rapamycin, by Merck Group (6 mentions)
Glutathione (gsh), by Merck Group (6 mentions)
Cycloheximide (chx), by Merck Group (6 mentions)
296 protocols using mitotempo
1
Protective Effects of PCr Against GS-Induced Toxicity
2
Phloroglucinol Protects ARPE-19 Cells from Oxidative Stress
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ARPE-19 cells (CRL-2302) were purchased from the American Type Culture Collection (Manassas, VA, USA) and routinely cultured in Dulbecco’s Modified Eagle Medium/F-12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (WELGENE Inc., Gyeongsan, Republic of Korea) as described previously [27 (link)]. To investigate beneficial effects of phloroglucinol on oxidative damage, cells were cultured in media containing desired concentrations of phloroglucinol and H2O2 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 24 h or pretreated with phloroglucinol, N-acetyl-L-cysteine (NAC), Mito-TEMPO, and/or 3-methyladenine (3-MA, Sigma-Aldrich Co., St. Louis, MO, USA) for 1 h prior to treatment with H2O2 for 24 h. To investigate the blocking effect of phloroglucinol on the generation of ROS induced by H2O2, cells were pretreated with phloroglucinol, NAC, and Mito-TEMPO for 1 h and then treated with H2O2 for 1 h. To acquire fluorescence images of ROS generation, γH2AX expression, and autophagic vacuoles, cells cultured on coverslips were stimulated with H2O2 in the presence or absence of phloroglucinol, NAC, and/or Mito-TEMPO. After treatment, cells were washed with phosphate-buffered saline and subjected to fluorescence staining.
3
Cytosolic Calcium Imaging in Rat Ventricular Myocytes
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To measure cytosolic Ca2+, cultured rat VMs were loaded with Fluo-3 AM (Invitrogen) at room temperature for 10 min in Ca2+-free Tyrode’s solution, followed by a 8 min wash in Tyrode’s solution containing 1 mmol/L Ca2+. Myocytes were perfused with Tyrode’s solution containing 1 mmol/L Ca2+ at room temperature during recordings. Fluo-3 AM was excited at 488 nm and fluorescence emission was collected at 500–550 nm wavelengths in line scan mode at 200 Hz sampling rate. The pacing protocol was followed as described above, with pacing for 5 min at 2 Hz, followed by the addition of either ISO (50 nmol/L) or ISO and low-dose caffeine (200 µmol/L), then further pacing for 5 min. For experiments with mitoTEMPO, VMs were preincubated with mitoTEMPO (20 μmol/L, Millipore Sigma) for 10 min prior to experimentation, and mitoTEMPO was included in the perfusion solution. Line scans were recorded during the last minute of pacing. Cytosolic Ca2+ transient amplitude is presented as ΔF/ΔF0, where F0 is basal fluorescence and ΔF = F–F0.
4
Intracellular Ca2+ Transient Measurement
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To measure intracellular Ca2+ transient, intact VMs were loaded with Fluo-3 AM (Invitrogen) at room temperature for 20 min in Tyrode’s solution containing 0.5 mmol/L Ca2+, followed by a 20 min wash in Tyrode’s solution containing 1 mmol/L Ca2+. Myocytes were then perfused with Tyrode’s solution containing 2 mmol/L Ca2+ and ISO (100 nmol/L) at room temperature during Ca2+ transient recordings. For experiments with mitoTEMPO, VMs were preincubated with mitoTEMPO (20 μmol/L, Millipore Sigma) for 20 min prior to experimentation, and mitoTEMPO was included in the perfusion solution. Fluo-3 AM was excited at 488 nm and fluorescence emission was collected at 500–550 nm wavelengths in line scan mode at 200 Hz sampling rate. To test for the propensity of triggered activity, VMs were stimulated for 10 s at 0.5 Hz and the latency between the last pacing stimulus and the subsequent SCW was calculated. To assess SR Ca2+ load, high-dose caffeine (10 mmol/L) was applied at the end of the experiments. The data are presented as ΔF/F0, where F0 is the basal fluorescence and ΔF = F–F0.
5
Measuring Mitochondrial ROS Using MitoSOX Red
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MitoSOX Red (Invitrogen Waltham, MA, United States) was used to measure mitochondrial ROS. A 5 mM stock solution prepared in DMSO was maintained frozen and diluted with water to a final 5 μM working concentration. This solution was used to cover sections of solid medium containing growing mycelia, during 20 min at 37°C. After this, the MitoSOX Red solution was removed, the mycelia rinsed two times with water and immediately observed using confocal microscopy. For Mito TEMPO/MitoSOX treatments, sections of solid medium containing growing mycelia were covered with a 100 μM solution of Mito TEMPO (Merck, KGaA, Darmstadt, Germany) during 2 h at 37°C. After this, the Mito TEMPO solution was removed, mycelia washed and then covered with a 5 μM MitoSOX Red solution during 20 min at 37°C and immediately observed using confocal microscopy. Images were processed using Software ZEN 2012 (Carl Zeiss, Jena, Germany).
6
Aβ oligomers' effect on HT-22 cells
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7
Mito-TEMPO and Mdivi-1 Modulate MSU-induced Inflammation
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C57BL/6 mice were intraperitoneally (ip) injected with Mito-TEMPO (20 mg/kg, Sigma–Aldrich, SML0737) or Mdivi-1(25 mg/kg, GLPBIO, GC10200), and after 1 h, the mice were injected with MSU suspension (3 mg in 100 μL PBS). After 6 h, the mice were sacrificed under CO2 anesthesia for extraction of peritoneal fluid. Peritoneal lavage fluid was collected to measure the IL-1β levels by ELISA and to assess lymphocyte recruitment by FCM using the leukocyte common antigen FITC-CD45 (BD BioScience, 553079) staining, Pacific blue-CD11b (Biolegend, USA, 101245) costaining with PE-F4/80 (BD BioScience, 565410), and APC-Ly-6G (Biolegend 127614), respectively.
The C57BL/6 mice were intraperitoneally injected with Mito-TEMPO (20 mg/kg, Sigma-Aldrich, SML0737) or Mdivi-1(25 mg/kg, GLPBIO, GC10200) and then injected the foot pad with MSU suspension (1 mg in 50 μL PBS). Twenty-four hours after injection with the MSU suspension, the swelling of the foot pad was measured with digital vernier caliper, and then the mice were sacrificed. Joint index evaluation was described previously [26 (link)].
The C57BL/6 mice were intraperitoneally injected with Mito-TEMPO (20 mg/kg, Sigma-Aldrich, SML0737) or Mdivi-1(25 mg/kg, GLPBIO, GC10200) and then injected the foot pad with MSU suspension (1 mg in 50 μL PBS). Twenty-four hours after injection with the MSU suspension, the swelling of the foot pad was measured with digital vernier caliper, and then the mice were sacrificed. Joint index evaluation was described previously [26 (link)].
8
Mito-TEMPO Ameliorates Sepsis-Induced Liver Injury
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To investigate the role of Mito-TEMPO in sepsis-induced liver damage, a lipopolysaccharide- (LPS-) induced experimental sepsis mouse model was established by injecting male mice intraperitoneally with LPS (E. coli 0111: B4, Sigma, USA) at a dosage of 5 mg/kg body weight. The control group was injected with the same volume of sterile phosphate-buffered saline (PBS), as in previous studies [26 (link), 27 (link)].
The mice were randomized and divided into three groups: the control group, the LPS group, and the LPS+Mito-TEMPO group (pretreatment with Mito-TEMPO followed by LPS injection). Mito-TEMPO (Sigma, USA) was intraperitoneally injected at a dose of 20 mg/kg body weight 1 h prior to LPS injection. The dosage of Mito-TEMPO is comparable with previous studies [28 (link), 29 (link)]. Animals were euthanized 24 h after LPS administration, and whole blood and liver samples were harvested for analysis.
The mice were randomized and divided into three groups: the control group, the LPS group, and the LPS+Mito-TEMPO group (pretreatment with Mito-TEMPO followed by LPS injection). Mito-TEMPO (Sigma, USA) was intraperitoneally injected at a dose of 20 mg/kg body weight 1 h prior to LPS injection. The dosage of Mito-TEMPO is comparable with previous studies [28 (link), 29 (link)]. Animals were euthanized 24 h after LPS administration, and whole blood and liver samples were harvested for analysis.
9
Chrysophanol Impacts Glioma Cell Dynamics
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In the experiments of effect of chrysophanol on glioma cells, cells were treated with different concentrations of chrysophanol (C15H10O4, ≥98%, 0, 10, 20, 50, 100 and 200 μM; 01542, Sigma-Aldrich, St. Louis, MO, USA) for 48 h. And during the researches on MitoTempo, cells were co-treated with 10 μM chrysophanol and 5 μM MitoTempo (5 μM; SML0737, Sigma-Aldrich, USA) for 24 h.
10
Mitigating Metabolic Dysfunction in Mice
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All procedures were performed in compliance with protocols approved by the University of Michigan Institutional Animal Care and Use Committee in accordance with NIH guidelines. All mice were purchased from Jackson Laboratory (Bar Harbor, ME). Male wild‐type, Sod2+/− (B6.129S7‐Sod2tm1Leb/J), Apoe−/− (B6.129P2‐Apoetm1Unc/J), and Apoe−/−/Sod2+/− mice were bred on the C57BL/6J background. Experimental mice were generated from breeding heterozygous mice, and genotypes were confirmed by polymerase chain reaction. Mice were housed in specific pathogen‐free rooms in ventilated cages at 22°C with 12‐hour light/dark cycles and free access to food and water. Animals (n=16 per group) were fed standard rodent chow for 1 (young) or 13 months (middle‐aged) and then switched to the Western diet (Harlan Teklad TD.88137; Envigo, Madison, WI) for 3 months.
Mice were treated with MitoTEMPO (Sigma‐Aldrich, St. Louis, MO) at 1500 μg/kg per day for 12 weeks as described previously.9 Briefly, at 13 months of age mice were randomly assigned to a MitoTEMPO or a vehicle treatment group (n=12). Microosmotic pumps (ALZET 1004; Durect, Cupertino, CA) were implanted subcutaneously under 2% inhaled isoflurane/O2 anesthesia. The pumps were replaced every 4 weeks. Mice were fed the Western diet for the duration of the experiment.
Mice were treated with MitoTEMPO (Sigma‐Aldrich, St. Louis, MO) at 1500 μg/kg per day for 12 weeks as described previously.
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