Primary hippocampal neurons were prepared from postnatal Sprague-Dawley rats (within 24 h; 6-8 neonates/experiment) obtained from the Center of Laboratory Animals of Anhui Medical University (Hefei, China) and placed in plates coated with poly-L-lysine (10 µg/ml). Neurobasal medium with B-27 supplement (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to culture the neurons at 37°C with 5% CO2 as described previously (21 (link)). The neurons were cultured for 7 days and divided into six groups: Control group, H2O2 (200 µM) group, H2O2 (200 µM) + Tempol (100 µM) group, and H2O2 (200 µM) + Rg1 (1, 5 and 10 µM) groups. With the exception of the control group, the neurons in the groups were treated with H2O2 (200 µM) and Tempol (100 µM) or Rg1 (1, 5 or 10 µM) for 24 h. Tempol (Santa Cruz Technology, Inc., Dallas, TX, USA) and Rg1 (content of Rg1 >98%; Chengdu Desite Biotechnology Co., Chengdu, China) were dissolved in distilled water and stored at −80°C. All experimental procedures were performed in accordance with the approved protocol of the Ethics Committee of Anhui Medical University (Anhui, China).
Tempol
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Tempol is a stable nitroxide free radical compound developed by Santa Cruz Biotechnology. It functions as a redox-active antioxidant and free radical scavenger.
Automatically generated - may contain errors
Lab products found in correlation
N acetylcysteine nac, by Merck Group (3 mentions)
Neurobasal medium with b 27 supplement, by Thermo Fisher Scientific (1 mentions)
Gemcitabine hydrochloride, by Merck Group (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Cisplatin, by Adipogen (1 mentions)
Z ietd fmk, by R&D Systems (1 mentions)
Z lehd fmk, by R&D Systems (1 mentions)
Z vdvad fmk, by R&D Systems (1 mentions)
N acetyl l cysteine, by Nacalai Tesque (1 mentions)
Necrostatin 1, by Santa Cruz Biotechnology (1 mentions)
Z vad fmk, by Enzo Life Sciences (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Clonogenic tumor cell isolation kit, by Cell Biolabs (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Allopurinol, by Merck Group (1 mentions)
Tiron, by Merck Group (1 mentions)
Resveratrol, by Merck Group (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
9 protocols using tempol
1
Neuroprotective Effects of Rg1 against H2O2
2
Inhibition of PI3K/AKT Pathway in Cancer
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The drugs employed were LY294002 (Cell Signaling Technology, Danvers, MA, USA) as an inhibitor of phosphatidylinositol 3-kinase (PI3K)-dependent AKT phosphorylation, N-acetylcysteine (NAC, Sigma-Aldrich), Tempol (Santa Cruz Biotechnology, Dallas, TX, USA), Cisplatin (AdipoGen, San Diego, CA, USA) and gemcitabine hydrochloride (Sigma-Aldrich). Final drug concentrations employed were 1.4 µM for LY294002, 1.25 mM for NAC, 1.25 mM for Tempol, 10 nM for Cisplatin and 10 nM for gemcitabine hydrochloride.
3
Inhibiting Apoptotic Pathways with Caspase Inhibitors
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For inhibition assays, the following inhibitors were added 1 h before the addition of TRAIL: pan-caspase inhibitor Z-VAD-FMK (Enzo Life Sciences, Farmingdale, NY, USA), caspase-8 inhibitor Z-IETD-FMK (R&D Systems, Minneapolis, MN, USA), caspase-9 inhibitor Z-LEHD-FMK (R&D Systems), and caspase-2 inhibitor Z-VDVAD-FMK (R&D Systems). N-acetyl-l -cysteine (NAC) was purchased from Nacalai Tesque. Tempol and necrostatin-1 were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA.
4
Isolation and Culture of Human Lung Cancer Cells
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Human lung cancer cells were freshly isolated from surgical tissues with Clonogenic Tumor Cell Isolation Kit (Cell Biolabs) according to the manual's instructions. Cells were cultured in complete RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (Gibco) supplemented with 2 mM glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin sulfate at 37°C under 5% CO2. Lipopolysaccharide (LPS) was purchased from Sigma. psiRNA vector expressing shRNA targeting human TLR4 gene (psirna42-htlr4) and control were purchased from Invivogen. Expression vector for human miR-21 (MI0000077) and control were purchased from Origene. Human miR-21 shRNA (shADV-215486) and control were purchased from Vector Biolabs. Tempol was purchased from Santa Cruz. All reagents were used according to manual's instructions.
5
Oxidative stress modulation in HT22 cells
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HT22 cells were seeded into six-well cell culture plates and divided into four groups: control, tempol alone (3 mM in media), BSO alone (1 mM in media), and BSO + tempol. BSO was purchased from Sigma-Aldrich (St. Louis, MO) and tempol was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Dose of BSO (1 mM) was selected based on our previously published data [16 (link)]. Cells were treated with BSO or vehicle (DMEM media) when 60–70% are confluent. tempol was added 10 h after addition of BSO. Cells were trypsinized at 14 h and used for various analyses. Thus, they are control (no treatment at 0 h, trypsinized at 14 h), BSO (1 mM BSO added at 0 h, trypsinized at 14 h), tempol (no treatment at 0 h, 3 mM tempol added at 10 h and trypsinized at 14 h), and BSO + tempol (1 mM BSO added at 0 h, 3 mM tempol added at 10 h and trypsinized at 14 h). All experiments were conducted at least 3-4 times.
6
Measuring Intracellular ROS Levels
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For determination of intracellular ROS levels, cells grown in 24-well plates were pre-incubated for 1 h with the fluorescent dye H2DCFDA (2’,7’-dichlorodihydrofluorescein diacetate, D-399, Thermo Fisher Scientific, Hennigsdorf, Germany, 10 µM), before starting treatment with effectors. After 2–24 h treatment, cells were harvested by trypsinization, washed several times with PBS, and analyzed by flow cytometry (FL1H). As a positive control, treatment with H2O2 (1 mM, 1 h) was applied. Different antioxidative treatments were used aiming at the suppression of celecoxib-induced ROS levels, including N-acetyl cysteine (NAC, Sigma-Aldrich, Taufkirchen, Germany, up to 4 mM), tocopherol (vitamin E, Fluka, Steinheim, Germany, up to 4 mM), glutathione (Sigma-Aldrich, 1 mM); Tiron (Sigma-Aldrich, 1 mM); 1400 W Sigma-Aldrich, 100 µM); Allopurinol (Sigma-Aldrich, 1 mM); TEMPOL (1-Oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine, Santa Cruz; 1 mM). Antioxidants were generally applied at 1 h before starting celecoxib treatment.
7
Evaluating Antioxidant Capacity in Cultured Cells
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Within this study, we used four different synthetic substances with AO capacity: resveratrol and NAC (Sigma-Aldrich), Tempol (Santa Cruz Biotechnologies) and DPI (Calbiochem). The concentrations applied to cells are listed in Table 1 . Synchronized cell cultures were treated with AOs either 2 hours (experimental scenario called quiescent cell AO treatment, «Q-AO-treatment») or 14 hours (experimental scenario called proliferating cell AO treatment, «P-AO-treatment») post serum stimulation and then cultivated as it has been previously described.
8
Triazole Cytotoxicity Assay with ROS Modulation
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Cytotoxic activity of triazoles and triazolium salts was determined by MTT assay22 (link), modified as described. Cells were seeded into 96-well tissue culture plates (3 x 103 cells/0.18 mL medium/well). The next day different concentrations of triazoles or triazolium salts were added (0.02 mL) to each well and each concentration was tested in quadruplicate. Following 72 h incubation at 37°C, the medium was aspirated, and 20 μg of the MTT dye (Sigma-Aldrich) /0.04 mL medium/well was added. Three hours later, formazan crystals were dissolved in DMSO (0.17 mL/well), the plates were mechanically agitated for 5 min and the optical density at 545 nm was determined on a microtiter plate reader (Awareness Technology Inc, Palm City, FL, USA). To examine the effect of reactive oxygen species (ROS) scavengers on survival of 2b treated cells, the same procedure was used as described above, except that two hours prior to addition of 2b, 5 mM of N-acetyl-cysteine (NAC, Sigma-Aldrich), or 1mM tempol (Santa Cruz Biotechnology, Dallas, TX, USA) was added in wells. Experiment was repeated at least three times.
9
Senescent ESCs Rejuvenation Cocktail
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Senescent ESCs were treated with 2 mM tempol (Santa Cruz Biotechnology, USA), 200 nM rapamycin (Calbiochem, USA), 5 mM metformin (Merk, Germany) for 7 days prior to decidualization induction. Cell culture medium supplemented with either compound was changed daily.
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