Mitochondria were isolated from cultured cell lines as previously described [21 (link)] with small modifications [22 (link)]. A mitochondrial pellet was immediately frozen in liquid nitrogen under anaerobic conditions. Then, lipids extraction and CoQ determination were performed as described previously [23 (link)]. Briefly, mitochondrial samples (0.6 mg of mitochondrial protein for each measurement) were lysed with 1% SDS and vortexed for 1 min. Then, a mixture of ethanol:2-propanol (95:5) was added and the samples vortexed again for 1 min. To recover CoQ, 5 mL of hexane was added, and the samples were centrifuged at 1000× g for 5 min at 4 °C. The upper phases from two extractions were recovered and dried using a rotary evaporator. Lipid extracts were suspended in 1 mL ethanol, dried in a speed-vac and stored at −20 °C. Samples were resuspended in a suitable volume of ethanol prior to HPLC injection. Lipid components were separated with an Alliance HPLC system (Waters) equipped with a 2707 autosampler and 2996 photodiode array detector, with HSST3 column (4.6 × 150 mm, 3.5 µm, Waters), preceded by a pre-column (4.6 × 20 mm, 3.5 µm, Waters), with a flow rate of 1 mL/min and a mobile phase containing 40:60 methanol/2-propanol. Commercial CoQ10 (Sigma) was used as an internal standard to detect the CoQ10 peak in the samples.
Coq10
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
CoQ10 is a naturally occurring compound found in the human body. It plays a key role in the production of cellular energy within the mitochondria of cells.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Taurine, by Merck Group (2 mentions)
Thymol, by Merck Group (1 mentions)
Sesamol, by Merck Group (1 mentions)
2 4 dichlorophenol, by Merck Group (1 mentions)
2 3 dihydroxybiphenyl, by Merck Group (1 mentions)
2 hydroxybiphenyl, by Merck Group (1 mentions)
Pre column, by Waters Corporation (1 mentions)
Alliance hplc system, by Waters Corporation (1 mentions)
Hss t3 column, by Waters Corporation (1 mentions)
V 1800, by Shimadzu (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Olive oil, by Merck Group (1 mentions)
Antimycin, by Merck Group (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
2 4 dinitrophenylhydrazine, by Merck Group (1 mentions)
57 protocols using coq10
1
Mitochondrial Coenzyme Q10 Determination
2
Encapsulation and Characterization of CoQ10 Nanoparticles
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Phospholipid nanoparticles solution (10%), in a form of nanospheres, was purchased from Nattermann Phospholipids (Germany). The encapsulation by CoQ10 (Sigma-Aldrich St. Louis, USA) at the concentration of 6 mg/ml, isolated after centrifugation at 6500 g for 30 min at 4 °C, was performed based on the method previously described.17 The efficacy of encapsulation was determined in a mixture of CoQ10-loaded liposomes and ethanol (3 ml) that was further vortexed for 3 min and centrifugated (2000 rpm, 5 min). The upper layer was separated and the absorbance was measured at 275 nm (V-1800 Shimadzu spectrophotometer). The encapsulation efficacy (%) was calculated as (amount of incorporated CoQ10)/(initial amount of added CoQ10) × 100.
3
Monosodium Glutamate and Coenzyme Q10 Effects
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All experiments were carried in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85–23, revised in 1996). Rats were divided randomly (10 rats/group) following the acclimatization period into: (1) control group: rats received 0.9% saline orally via gavage, and olive oil via intra-peritoneal injection, (2) MSG-treated group: rats received single daily dose (5 g/kg body weight dissolved in 0.9% saline via oral gavage) of monosodium glutamate (MSG, Sigma-Aldrich, UK) for 15 consecutive days, (3) MSG and olive oil (OO)-treated group: rats received MSG (5 g/kg body weight dissolved in 0.9% saline via oral gavage), and OO via intra-peritoneal injection (IP) for 15 consecutive days, and (4) MSG and CoQ10-treated group: rats received MSG (5 g/kg body weight dissolved in 0.9% saline via oral gavage), and CoQ10 (Sigma-Aldrich, UK, 10 mg/kg body weight dissolved in olive oil IP) daily for 15 consecutive days.
4
In vivo CoQ10 repletion in mice
5
Coenzyme Q10 and Rosuvastatin Protocol
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CoQ10 was obtained from Sigma-Aldrich (St. Louis, MO, United States), whereas rosuvastatin (RUS) and CoQ10 solubilizing agents that include [Labrasol, Labrafil (M1944 CS), and Capryol 90] were generously gifted from the Global Napi for Pharmaceutical industry (Cairo, Egypt) and Gattefosse (Lyon, France), respectively.
6
Mitochondrial Delivery of CoQ10 Nanoparticles
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Cells were pretreated with
free CoQ10 (Sigma, USA), nontargeted nanoparticles loaded
with CoQ10 (NT-CoQ10-NPs), or mitochondria-targeted
nanoparticles loaded with CoQ10 (T-CoQ10-NPs)
for 24 h, followed by antiretroviral treatment for different time
points as specified in the respective experiments. The same dose of
CoQ10 of 500 nM, which was determined to be optimal with
preliminary experiments, was used in all experiments to compare the
efficiency of mitochondrial delivery. The stock solution of synthesized
NPs was in micromolar concentrations that were diluted to the working
concentration of 500 nM CoQ10 by adding it to cell culture
media.
free CoQ10 (Sigma, USA), nontargeted nanoparticles loaded
with CoQ10 (NT-CoQ10-NPs), or mitochondria-targeted
nanoparticles loaded with CoQ10 (T-CoQ10-NPs)
for 24 h, followed by antiretroviral treatment for different time
points as specified in the respective experiments. The same dose of
CoQ10 of 500 nM, which was determined to be optimal with
preliminary experiments, was used in all experiments to compare the
efficiency of mitochondrial delivery. The stock solution of synthesized
NPs was in micromolar concentrations that were diluted to the working
concentration of 500 nM CoQ10 by adding it to cell culture
media.
7
Fibroblast Culture with CoQ10 Treatment
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The fibroblasts were cultured in DMEM 1885 medium supplemented with 1% Glutamax, 10% FBS (In Vitro) and 1% penicillin/streptomycin at 37°C, 5% CO2 95% relative humidity. Cells treated with CoQ10 were incubated in standard medium containing 10 µmol/l CoQ10 (Sigma-Aldrich) for 7 days before harvest/measurements.
8
Preparation and characterization of antioxidant compounds
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Manganese (Mn) as manganese (II) chloride tetrahydrate (MnCl2.4H2O), was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was freshly dissolved in normal saline (NaCl; 0.9%, “El-Nasr”) [68 (link)]. Sesamol, thymol, and CoQ10 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were suspended in 1% tween 80 in normal saline [63 ,64 (link),65 (link)]. Wheatgrass (WG) was purchased from Bioglan superfoods, UK and was freshly prepared, suspended in 1% tween in normal saline. The chemical constituents of WG were previously identified and analyzed using high-performance liquid chromatography (HPLC) in our previous work as described by [66 (link)]. All other chemicals and solvents used in the current study were of the highest grade commercially available.
9
Arabidopsis Coenzyme Q Biosynthesis
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All A. thaliana lines used are in the Columbia-0 (Col-0) background. The T-DNA insertional mutants of SALK_073461 and emb2421 were obtained from the Nottingham Arabidopsis Stock Center. Primers used in this investigation are listed in table S4.
Arabidopsis and N. benthamiana plants were grown in greenhouse at 22°C under long-day condition (16 hours light and 8 hours dark). CoQ4, CoQ6, CoQ8, CoQ9, CoQ10, 2,4-dichlorophenol, 3,5-dichlorocatechol, 2-hydroxybiphenyl, and 2,3-dihydroxybiphenyl standards were purchased from Sigma-Aldrich.
Arabidopsis and N. benthamiana plants were grown in greenhouse at 22°C under long-day condition (16 hours light and 8 hours dark). CoQ4, CoQ6, CoQ8, CoQ9, CoQ10, 2,4-dichlorophenol, 3,5-dichlorocatechol, 2-hydroxybiphenyl, and 2,3-dihydroxybiphenyl standards were purchased from Sigma-Aldrich.
10
CoQ10 Deficiency Cell Culture Protocol
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HEK293, HeLa cells and human fibroblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine, 75 U/mL penicillin, 50 µg/mL streptomycin (Invitrogen) at 37 °C in a 5% CO2 incubator. Fibroblasts of patients with CoQ10 deficiency were previously described38 (link),51 (link). They had been obtained with the informed consent of the parents of the patients. The study was reviewed by the local ethical committee (Comitato Etico per la Sperimentazione Clinica della Provincia di Padova) – protocol AOPD2019 #0020044). All experiments were performed in accordance with the relevant guidelines and regulations of the University of Padova.
HEK293 COQ4−/− were generated in our laboratory as described40 (link) and were cultured in the presence of pyruvate (1 mM) and uridine (50 μg/mL) with 4% FBS, to minimize CoQ10 content in the medium.
For MRC activity and ATP measurement, cells were cultured for 2 days in DMEM medium containing low glucose (2 mM), to force mitochondrial respiration.
CoQ10, MK-4, CoQ4 and DUB were purchased from Sigma (Milan, Italy). They were dissolved in ethanol and added to the cells as less than 1% of total cell culture volume.
HEK293 COQ4−/− were generated in our laboratory as described40 (link) and were cultured in the presence of pyruvate (1 mM) and uridine (50 μg/mL) with 4% FBS, to minimize CoQ10 content in the medium.
For MRC activity and ATP measurement, cells were cultured for 2 days in DMEM medium containing low glucose (2 mM), to force mitochondrial respiration.
CoQ10, MK-4, CoQ4 and DUB were purchased from Sigma (Milan, Italy). They were dissolved in ethanol and added to the cells as less than 1% of total cell culture volume.
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