3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt

Manufactured by Thermo Fisher Scientific

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a chemical compound used as a colorimetric assay for assessing cell metabolic activity. It is a water-soluble tetrazolium salt that can be reduced to an insoluble purple formazan product by the action of dehydrogenase enzymes, reflecting the number of viable cells present.

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197 protocols using 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt

Cell Viability and Apoptosis Assays

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Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Bovine Serum Albumin (BSA) and Trypsin (0.5% EDTA) were obtained from GIBCO^TM, (Invitrogen Corporation). Artemisinin, Penicillin/Streptomycin, Lipofectamine^R 2000 reagent (Invitrogen Co.,CA, USA), DMSO, H₂O₂ were obtained from Sigma Aldrich (St. Louis, MO, USA). Sodium Azide (NaN₃) were obtained from Acros Organic, (New Jersey, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Cell ROXs Deep Red Reagent, and Hoechst 33342 were purchased from Molecular Probes (Eugene, or, USA). Pierce BCA protein assay kit and Halt^TM Protease and phosphatase inhibitor cocktail were purchased from Thermo Scientific USA, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanineiodide (JC-1) from Beyotime, Annexin V-FITC/PI were purchased from BBI Life Sciences. anti-p-AMPK, anti-AMPK and anti-β-Actin antibodies were purchased from Cell Signaling Technology (CST) (Beverly, MA, USA). Anti-Rabbit IgG HRP-conjugated secondary antibody was purchased from Promega (Madison, Wl, USA).

Li S., chaudhary S.C., Zhao X., Gaur U., Fang J., Yan F, & Zheng W. (2019). Artemisinin Protects Human Retinal Pigmented Epithelial Cells Against Hydrogen Peroxide-induced Oxidative Damage by Enhancing the Activation of AMP-active Protein Kinase. International Journal of Biological Sciences, 15(9), 2016-2028.

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Enzymatic Assay for Cell Proliferation

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The pepsin and pancreatin enzymes were purchased from Nacalai Tesque Inc. (Kyoto, Japan). The chicken EGF ELISA kit was purchased from Cusabio Technology LLC (Houston, USA). Sodium hydrogen carbonate, sodium bicarbonate, hydrochloric acid, sodium hydroxide, foetal bovine serum (FBS), and biological-grade dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, USA). Roswell Park Memorial Institute (RPMI) 1640 medium with L-glutamine, 2.5% trypsin (10x), and 10,000 U/mL penicillin–10 mg/mL streptomycin were purchased from GIBCO (New York, USA). Phosphate-buffered saline (PBS) was from Life Technologies (Maryland, USA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Molecular Probes (Oregon, USA). Human EGF (hEGF) was purchased from Pepro Tech Inc (New Jersey, USA).

Tan S.N., Sani D., Lim C.W., Ideris A., Stanslas J, & Lim C.T. (2020). Proximate Analysis and Safety Profile of Farmed Edible Bird's Nest in Malaysia and Its Effect on Cancer Cells. Evidence-based Complementary and Alternative Medicine : eCAM, 2020, 8068797.

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Mangostin and Memantine Cytoprotective Effects

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alpha-Mangostin (≥98% purity) and memantine hydrochloride (≥98%) were purchased from Sigma Aldrich (St Louis, MO, USA). Hydrogen peroxide (H₂O₂, 30%), Annexin V & Dead Cell Assay Kit, and Muse™ Caspase-3/7 Kit were obtained from Merck Millipore (Darmstadt, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2′,7′-dichlorodihydrofluorescin diacetate (H₂DCFDA) were purchased from Molecular Probes (Eugene, Oregon, USA). Monoclonal antibodies against BAX, BCL-2, SIRT1, SIRT3, FOXO3a, CAT, SOD2, and actin, as well as horseradish peroxidase (HRP)-conjugated goat anti-mouse and rabbit secondary antibodies, were purchased from Cell Signaling Technology (Beverly, MA, USA). Enhanced chemiluminescence (ECL) Plus Western blotting reagent was supplied from Amersham Biosciences (Piscataway, NJ, USA). All other chemicals and reagents were of analytical grade from Sigma Aldrich (St Louis, MO, USA).

Ruankham W., Suwanjang W., Phopin K., Songtawee N., Prachayasittikul V, & Prachayasittikul S. (2022). Modulatory Effects of Alpha-Mangostin Mediated by SIRT1/3-FOXO3a Pathway in Oxidative Stress-Induced Neuronal Cells. Frontiers in Nutrition, 8, 714463.

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Synthesis and Evaluation of Cationic Polymers for Gene Delivery

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All chemicals were purchased from Sigma Aldrich
and used without further purification unless specified otherwise. N-(2-aminoethyl) methacrylamide hydrochloride (AEMA·HCl)
and N-[3-(N,N-dimethylamino)propyl]methacrylamide
(DMAPMA) were purchased from Polyscience, Inc. (Warrington, PA), and
DMAPMA was purified via vacuum distillation prior to use. 4,4′-Azobis(4-cyanovaleric
acid) (V-501) was recrystallized from methanol. 2-deoxy-2-methacrylamido
glucopyranose (MAG)³⁵ and the chain transfer
agent (CTA) 4-cyano-4-(propylsulfanylthiocarbonyl) sulfanylpentanoic
acid (CPP)²⁹ were synthesized according
to previous reports. Cell culture media, Dulbecco’s Modified
Eagle Medium (DMEM) and Opti-MEM, antibiotic/antimycotic, fetal bovine
serum (FBS), phosphate-buffered saline (PBS), and nuclease-free water
were purchased from Gibco (Carlsbad, CA). HeLa (Human cervix adenocarcinoma)
cells and HepG2 (human liver hepatocellular carcinoma) cells were
purchased from ATCC (Rockville, MD). JetPEI was purchased from Polyplus-Transfection
Inc. (Illkirch, France). Glycofect was provided by Techulon (Blacksburg,
VA) as a gift. 3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium
bromide (MTT) was purchased from Molecular Probes (Eugene, Oregon).
Luciferase assay kits were purchased from Promega Corp. (Fitchburg,
WI). Protein assay kits were purchased from Bio-Rad Laboratories (Hercules,
CA).

Wu Y., Wang M., Sprouse D., Smith A.E, & Reineke T.M. (2014). Glucose-Containing Diblock Polycations Exhibit Molecular Weight, Charge, and Cell-Type Dependence for pDNA Delivery. Biomacromolecules, 15(5), 1716-1726.

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Regulation of Endothelial Nitric Oxide Synthase

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Ponatinib was purchased from Selleckchem (Houston, TX). VEGFR tyrosine kinase inhibitor II (VRI) was purchased from Calbiochem (Darmstadt, Germany). 1-phenyl 2-thiourea (PTU) was obtained from Sigma-Aldrich (St. Louis, MO). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 4-amino-5-methylamino-2’,7’-difluorofluorescein (DAF-FM) were purchased from Molecular Probes (Eugene, OR). Medium 200 was purchased from Gibco Invitrogen (Carlsbad, CA). Primary antibodies for phospho-eNOS (Ser1179), total-eNOS, phospho-Akt (Ser473), total Akt, phospho-p38 (Thr180/Tyr182), total p38, phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, beta-actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Ai N., Chong C.M., Chen W., Hu Z., Su H., Chen G., Lei Wong Q.W, & Ge W. (2018). Ponatinib exerts anti-angiogenic effects in the zebrafish and human umbilical vein endothelial cells via blocking VEGFR signaling pathway. Oncotarget, 9(62), 31958-31970.

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Apoptosis Induction and Oxidative Stress Assay

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Roswell Park Memorial Institute (RPMI) 1640, penicillin, fetal bovine serum (FBS), and streptomycin were purchased from Life Technologies Inc. (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Molecular Probes Inc. (Eugene, OR, USA). Propidium iodide (PI), N-acetyl-L-cysteine (NAC), and 2-mercaptoethanol were acquired from Sigma Chemical (St. Louis, MO, USA). Phenylmethylsulfonylfluoride (PMSF) and annexin V-fluorescein isothiocyanate (FITC) were purchased from BD Biosciences (San Jose, CA, USA). Dichlorofluorescein diacetate (DCFH-DA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All inhibitors for caspases were purchased from Calbiochem (Bad Soden, Germany). Enhanced chemiluminescence (ECL) reagent was obtained from EMD Millipore (Billerica, MA, USA). Tris-buffered saline was obtained from Boster Biological Technology Ltd. (Wuhan, China). Caspase-3 and caspase-9 antibodies were procured from Cell Signaling Technology (Beverly, MA, USA). Caspase-8 and β-actin antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were purchased from The Jackson Laboratory (West Grove, PA, USA).

Wang Y.Y., Kwak J.H., Lee K.T., Deyou T., Jang Y.P, & Choi J.H. (2020). Isoflavones Isolated from the Seeds of Millettia ferruginea Induced Apoptotic Cell Death in Human Ovarian Cancer Cells. Molecules, 25(1), 207.

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Cell Signaling Pathway Assay Protocol

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Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium, Medium 199 (M199), Opti-modified Eagle’s medium (Opti-MEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). Phosphate buffered saline (PBS), DMSO, RNase A, leupeptin, aprotinin and phenylmethylsulfonylfluoride (PMSF) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Antibodies against anti-phospho-Akt, anti-phospho-IkBα and anti-phospho-IKKα/β were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies against anti-Akt, anti-p65, anti-IkBα, anti-IKKα/β, anti-PARP and anti-ß-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CellTracker^TM was obtained from Invitrogen (Grand Island, NY, USA).

Ahn J.H., Choi J.M., Kang E.S., Yoo J.H., Cho Y.J., Jang D.S, & Choi J.H. (2022). The Anti-Endometriotic Effect of Cyperi Rhizoma Extract, Inhibiting Cell Adhesion and the Expression of Pain-Related Factors through Akt and NF-kB Pathways. Medicina, 58(3), 335.

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Hederagenin and CORT Mechanism Study

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Hederagenin (≧98% purity by HPLC) was purchased from Nanjing Puyi Biological Biotech Co. Ltd. (Nanjing, China). CORT (purity ≧98%) was purchased from Meilunbio (Dalian, China). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium, penicillin and streptomycin were purchased from Gibco (Grand Island, United States). Primary antibodies for GAPDH, anti-phospho-AKT, anti-AKT, anti-phospho-FoxO3a, anti-FoxO3a, anti-phosphor-GSK3β and anti-GSK3β were purchased from Cell Signaling (CST, United States), Brain-derived neurotrophic factor (BDNF) was purchased from Abcam (Cambridge, United Kingdom). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich Co. (St Louis, United States). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Molecular Probes (Eugene, OR, USA). LY294002 was purchased from Calbiochem. MK-2206 was purchased from Selleckchem. Annexin V FITC Apoptosis Detection Kit was obtained from BD Biosciences (San Diego, CA) The commercial kit for measuring Reactive Oxygen Species (ROS) and mitochondrial membrane potential ∆Ψm (JC-1) content were purchased from Beyotime Biotechnology (Shanghai, China).

Lin R., Liu L., Silva M., Fang J., Zhou Z., Wang H., Xu J., Li T, & Zheng W. (2021). Hederagenin Protects PC12 Cells Against Corticosterone-Induced Injury by the Activation of the PI3K/AKT Pathway. Frontiers in Pharmacology, 12, 712876.

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Cytotoxicity Evaluation of Anticancer Agents

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Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI-1640) medium, minimum Eagle's medium (MEM), fetal bovine serum (FBS), and 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) were obtained from Life Technologies INC. (Grand Island, NY, USA). Trypan blue, penicillin, streptomycin, dimethyl sulfoxide (DMSO), VP-16 (Etoposide) and taxol were supplied by Sigma Chemical Co. (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Hydroxypopyl-β-cyclodextrin (HP-β-CD) was obtained from Nihon Shoukuhin Kako Co. Ltd. (Shibuya-ku, Tokyo, Japan). Phosphate buffer saline (PBS), annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit, and caspase activity kit were purchased from Keygentec (Nanjing, Jiangsu, China). The water used in the experiments was obtained from the Milli-Q Water Purification System (MilliporeCorp., Billerica, MA, USA).

Ma R., Chen J.T., Ji X.Y., Xu X.L, & Mu Q. (2020). Hydroxypropyl-β-Cyclodextrin Complexes of Styryllactones Enhance the Anti-Tumor Effect in SW1116 Cell Line. Frontiers in Pharmacology, 11, 484.

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Evaluating Anticancer Pathways

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Camptothecin, doxorubicin hydrochloride, etoposide, and hydrogen peroxide (H₂O₂) were purchased from Sigma (St. Louis, Missouri). F-12K Medium was supplied by Gibco (Bethesda, Maryland). Fetal bovine serum (FBS) and horse serum were obtained from Invitrogen (Carlsbad, California). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Molecular Probes (Eugene, Oregon). Primary antibodies against phosphorylated phosphoinositide 3-kinase (p-PI3K; p-FAK and Tyr925), phosphorylated Akt (p-Akt; Ser473), phosphorylated mammalian target of rapamycin (p-mTOR), phosphatase with tensin homology (PTEN), heme oxygenase-1 (HO-1), nuclear factor-E2-related factor 2 (Nrf2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and secondary antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts). Hoechst 33342 staining kit and LY294002 were purchased from Jiangsu Beyotime Institute of Biotechnology. The enhanced chemiluminescence (ECL) detection kit was purchased from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). All other chemicals of analytical grade were purchased from local sources.

Zhang C., Chen S., Bao J., Zhang Y., Huang B., Jia X., Chen M., Wan J.B., Su H., Wang Y, & He C. (2015). Low Doses of Camptothecin Induced Hormetic and Neuroprotective Effects in PC12 Cells. Dose-Response, 13(2), 1559325815592606.

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