Human aortic endothelial cells (HAECs) and human THP-1 monocytic cells were used in this study. HAEC cells were purchased from Lonza (Basel, Switzerland) and cultured in endothelial growth media EBM-2 recommended by the manufacturer (Lonza, Switzerland). THP-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 (Gibco, Waltham, MA, USA). All cells were maintained at 37°C in a 5% CO2 atmosphere. Oxidized LDL (40 μg/mL, Acesar, Waltham, MA, USA) were exposed on each cell for 24 hours to 48 hours. NQDI-1 (ASK1 inhibitor, Tocris Bioscience, Bristol, UK) was dissolved in DMSO and treated on each cell with 600 nM to inhibit ASK1 activation at 4 hours before oxLDL exposure. For the masking receptor, CD36, neutralizing the CD36 antibody (FA6-152, Abcam, Cambridge, MA, USA), was pretreated on each cell (2 μg/mL) 4 hours before oxLDL exposure. To restore ASK1, the peptide for ASK1 was synthesized to contain the active sites (Thr845) of ASK1 from amino acids 836–875.
Nqdi 1
Manufactured by Bio-Techne
Sourced in United Kingdom
NQDI-1 is a small molecule inhibitor that selectively inhibits the NQO1 enzyme. NQO1 is a quinone reductase involved in cellular redox processes. NQDI-1 provides a tool to study the biological functions of NQO1.
Automatically generated - may contain errors
Lab products found in correlation
Thp 1 cell, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Oxldl, by Bio-Techne (1 mentions)
Fa6 152, by Abcam (1 mentions)
Ox ldl, by Abcam (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Ebm 2, by Lonza (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Penicillin streptomycin, by Cytiva (1 mentions)
Rpmi 1640 medium, by Cytiva (1 mentions)
Resveratrol, by Merck Group (1 mentions)
L glutamine, by Cytiva (1 mentions)
Insulin, by Fujifilm (1 mentions)
Sh sy5y neuroblastoma cells, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
8 protocols using nqdi 1
1
Modulation of Endothelial-Monocyte Interactions
2
Hypoxia-Reoxygenation Endothelial Cell Assay
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Before OGD, the culture medium was discarded, washed with phosphate buffer saline (PBS) and changed with deoxygenated glucose-free balanced salt solution (BSS) under the anaerobic chamber (Forma Scientific, Inc., Marietta, GA, USA). Endothelial cells were exposed to OGD condition for 6 h. After hypoxia, BSS solution was discarded and changed into DMEM cultured media and cells were transferred to a CO2 incubator for 24 h. After reperfusion, cells and EC-CM were collected and stored at -80°C. NQDI-1 (600 nM, Tocris Bioscience, Bristol, UK), an inhibitor of ASK1, was treated in cultured media from 1 h before hypoxia to 6 h during hypoxia.
3
Differentiation and Activation of THP-1 Macrophages
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Human monocyte leukemia cells (THP-1 cells) (ATCC, Manassas, VA, USA) were cultured at 37℃ with 5% CO2 in RPMI 1640 medium (Hyclone Laboratories, South Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone Laboratories), 2 mM of L-glutamine, and 100 units/ml of penicillin/streptomycin (Hyclone Laboratories). For differentiation to adherent THP-1 derived macrophages, cells were plated at 1×106 cells/plate with 5 ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) for 48 hrs [34 (link)], followed by stimulation with 1 µg/ml of lipopolysaccharides (LPS) (Sigma-Aldrich) for 4 hrs. THP-1 cells were treated with resveratrol (Sigma-Aldrich) at various concentrations (25, 50, 100, and 200 µM). NQDI-1, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, was purchased from Tocris Bioscience (TOCRIS Bioscience, Bristol, UK). THP-1 cells were pretreated with ASK1 inhibitor (600 nM) for 2 hrs to inhibit ASK1 activation.
4
NQDI 1 Inhibits CS-Induced Lung Damage
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A dosage of 10 mg/kg bodyweight of NQDI 1 (Tocris Bioscience, Bristol, UK) or an equal volume of vehicle was intraperitoneally (i.p.) administered into mice after CS instillation (10 mice per group). Detailed methods have been outlined in the previous article (17 (link)). Lung tissues were harvested and used for immunohistochemical and western blot analyses after 7- and 56-days exposure to CS.
5
Differentiation and Insulin Stimulation of SH-SY5Y Neuroblastoma Cells
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SH-SY5Y neuroblastoma cells (ATCC, Manassas, VA, USA) are capable of differentiating into neuron-like cells in the presence of retinoic acid (RA). Undifferentiated SH-SY5Y cells were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 100 μg/mL of penicillin-streptomycin (Gibco). SH-SY5Y cells were passaged at least twice, and differentiated with a replacement of fresh media supplemented with 1% FBS and 5 μM of RA, and cultured in DMEM media with 100 nM of insulin (Wako Chemicals, Richmond, VA, USA) for 3 days, with a replacement of fresh medium every 24 h [17 (link)]. Afterwards, medium was replaced with serum-free DMEM media. After 30 min, cells were exposed to 1 μM of insulin for 15 min [18 (link),19 (link)]. NQDI-1, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, was purchased from Tocris Bioscience (Bristol, UK). Cells were pretreated with ASK1 inhibitor (600 nM) for 2 h to inhibit ASK1 activation before IR stress.
6
Modulation of miR-Let7A in BV2 Microglia
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BV2 microglia were treated with miR-Let7A mimic or anti-miR-Let7A. Let-7A mimic (Let-7A precursor molecules are considered to be processed into mature miRNA by mimicking the Let-7A natural shearing process) and anti-Let-7A (Let7A inhibitor) were purchased from Ambion (Austin, TX, USA). For the transfection of RNA duplexes, a 20 nM final concentration of Let-7A miRNA mimic or Let-7A inhibitor was mixed with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in Opti-MEM medium and incubated at room temperature for 10 min. The mixture was added to cells in 6-well plates. After cells reached 60% confluence, they were harvested for total protein or RNA extraction. ASK1 inhibitor (NQDI-1) was purchased from Tocris Bioscience (Bristol, UK). BV2 microglial cells were pretreated with ASK1 inhibitor (600 nM) to inhibit ASK1 activation 2 h before high-glucose injury.
7
Cell Culture and Stress Treatments
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Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM
(Thermo Fisher, Waltham, MA, USA)) containing 100 units/ml penicillin,
100 μg/ml streptomycin (Cellgro, Manassas, VA, USA),
5% fetal bovine serum (FBS, (CellGro)), and 5% fetal calf serum
(Gemini, West Sacramento, CA, USA) in a 5% CO2 incubator at
37 °C. The following treatment concentrations were used:
H2O2 ((Thermo Fisher) 200 nM), Staur (Staur
(Sigma, St. Louis, MO, USA), 1 nM), ASK1 inhibitor (NQDI-1 (Tocris,
Bristol, UK), at 10 μM, 30 μM, and
50 μM).
(Thermo Fisher, Waltham, MA, USA)) containing 100 units/ml penicillin,
100 μg/ml streptomycin (Cellgro, Manassas, VA, USA),
5% fetal bovine serum (FBS, (CellGro)), and 5% fetal calf serum
(Gemini, West Sacramento, CA, USA) in a 5% CO2 incubator at
37 °C. The following treatment concentrations were used:
H2O2 ((Thermo Fisher) 200 nM), Staur (Staur
(Sigma, St. Louis, MO, USA), 1 nM), ASK1 inhibitor (NQDI-1 (Tocris,
Bristol, UK), at 10 μM, 30 μM, and
50 μM).
8
Cytotoxicity Evaluation of Therapeutic Compounds
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L-glutamine, fetal bovine serum, penicillin, streptomycin, amphotericin B, Hanks’ salts, Earle’s salts, trypsin, DMEM, MEM, and (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from PanEco, Moscow, Russia. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), isopropanol, HCl, CHAPS, protease inhibitor cocktail, EDTA, dithiothreitol, HEPES, DMSO, SCP0139, toluene, acetonitrile, carbon tetrachloride, diethylenetriamine, urea, triethylamine, 4-chlorophenyl isocyanate, 3,4-dichlorophenyl isocyanate, and D-glucose were from Sigma-Aldrich, St. Louis, MO, USA. Hydroxychloroquine, Z-VAD-FMK, necrostatin-1, necrosulfonate, NQDI-1, Ac-DEVD-AFC, and Ac-LEHD-AFC were from Tocris Bioscience, Bristol, UK. The apoptosis assay kit was from Abcam, Cambridge, MA, USA.
Cell lines were purchased from ATCC, Manassas, VA, USA.
Cell lines were purchased from ATCC, Manassas, VA, USA.
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