Melatonin, streptozotocin (STZ), and 2,3,5-triphenyl tetrazolium chloride (TTC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). LY294002 and 3-TYP were purchased from MedChemExpress (MCE; Monmouth Junction, NJ, USA). Dulbecco’s Modified Eagle Medium (DMEM) and penicillin/streptomycin were obtained from GENOM (Hangzhou, China), fetal bovine serum (FBS) were bought from TIANHANG (Zhejiang, China). The mitochondrial membrane potential assay kit with JC-1 and the ROS Assay Kit were got from Beyotime (Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Absin (Shanghai, China). Kits for detecting malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, and ATP content and Mitochondria Isolation Kit were purchased from the Nanjing Jiancheng (Nanjing, Jiangsu, China). The Annexin V-FITC Apoptosis Detection Kit was obtained from KeyGEN (Nanjing, Jiangsu, China). Primary antibodies against p-Akt, Akt, cytochrome c (cyto-C), and β-actin, as well as the secondary antibodies, were all purchased from Cell Signaling Technology (CST; Boston, MA, USA). Primary antibodies against SIRT3, SOD2 (acetyl K68) (ac-SOD2), SOD2, TFAM, TFAM were purchased from Abcam (Cambridge, MA, USA).
Mitochondria isolation kit
Manufactured by Nanjing Jiancheng
Sourced in China
The Mitochondria Isolation Kit is a laboratory equipment designed to extract and purify mitochondria from various types of cells and tissues. The kit includes reagents and protocols for the efficient separation and isolation of mitochondria, which are essential organelles responsible for energy production within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Melatonin, by Merck Group (1 mentions)
3 typ, by MedChemExpress (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Ly294002, by MedChemExpress (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Cell counting kit 8 cck 8, by Absin (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
1 plus kit, by Hypoxyprobe (1 mentions)
4 protocols using mitochondria isolation kit
1
Melatonin Mitigates Oxidative Stress-Induced Apoptosis
2
Retinal Mitochondria Isolation
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Cytosolic and mitochondrial fractions were isolated using fresh retinal tissues and a mitochondria isolation kit (cat#G006-1-1, Nanjing Jiancheng Bioengineering Institute) per the manufacturer's instructions.
3
Mitochondrial Function Regulation by Rg5 and Metformin
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Differentiated adipocytes were cultured in 15 cm diameter plates and treated with Rg5 (10 μM) or metformin (1 mM) for 4 h, and the cells were collected after PBS wash. Mitochondria were isolated by Mitochondria Isolation Kit (Jiancheng, Nanjing, China). Then, 10 μg of protein was collected for the assay of complex I or IV activity with commercial kit (Jiancheng, Nanjing, China). For ATP measurement, 3T3-L1 cells were treated with regents for 4 h, the cells extracted with lysis buffer, and centrifuged at 12 000 g for 15 min at 4°C. After quantification, the supernatants were detected by ATP Assay Kit (Beyotime, Nanjing, China).
4
Hepatocyte Metabolism and Mitochondrial Function
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The primary mouse hepatocytes were maintained in DMEM with 10% FBS. After planting, the media were replaced with serum-free DMEM for 18 h. After washing, the cells were incubated in glucose-free media supplemented with 10 mmol/l pyruvate, 100 nmol/l glucagon and 100 μmol/l palmitate alone or with Rg5 at given concentrations for 6 h. The supernatant fraction was then collected for glucose analysis.
Detection of mitochondrial complex II activity The primary mouse hepatocytes were treated with Rg5 10 μmol/l in the presence of palmitate for 8 h, and mitochondria were isolated using a mitochondria isolation kit (Jiancheng). Protein 10 μg was then collected to assay complex II activity using a commercial kit (Jiancheng).
Pimonidazole staining for hypoxia and histopathological examination The HFD-fed mice were injected i.p. with pimonidazole 60 mg/kg. The isolated liver was dehydrated and embedded in paraffin and pimonidazole staining was performed using a Hypoxyprobe-1 Plus Kit (Hypoxyprobe, Burlington, MA, USA). For hypoxia detection in the primary mouse hepatocytes, pimonidazole 200 μmol/l was added to the cells before incubation with palmitate 100 μmol/l. The image was visualised using a fluorescence microscope. Liver slices were stained with haematoxylin and eosin, according to standard protocols.
Detection of mitochondrial complex II activity The primary mouse hepatocytes were treated with Rg5 10 μmol/l in the presence of palmitate for 8 h, and mitochondria were isolated using a mitochondria isolation kit (Jiancheng). Protein 10 μg was then collected to assay complex II activity using a commercial kit (Jiancheng).
Pimonidazole staining for hypoxia and histopathological examination The HFD-fed mice were injected i.p. with pimonidazole 60 mg/kg. The isolated liver was dehydrated and embedded in paraffin and pimonidazole staining was performed using a Hypoxyprobe-1 Plus Kit (Hypoxyprobe, Burlington, MA, USA). For hypoxia detection in the primary mouse hepatocytes, pimonidazole 200 μmol/l was added to the cells before incubation with palmitate 100 μmol/l. The image was visualised using a fluorescence microscope. Liver slices were stained with haematoxylin and eosin, according to standard protocols.
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