PC3 cells (3000–5000 cells/well) were seeded onto a 96-well plate. After one day, freshly prepared RPMI media containing different concentrations (0, 1, 5, 10 µM; n = 5) of FTY720 (Selleckchem, Houston, TX, USA) or compound 10 (0, 1, 3, 15, 30 µM; n = 5) were added to the cells, and the mixtures were incubated for 48 or 72 h. The relative number of viable cells was determined by an assay using 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Saint-Louis, MO, USA), according to the manufacturer’s instructions, on a microplate reader at OD 570 nm. The experiment was repeated in triplicate, and data were represented as the mean ± standard deviation (s.d).
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt
Manufactured by Merck Group
Sourced in United States, Germany, China, Italy, Sao Tome and Principe, Macao, United Kingdom, Spain, Poland, Switzerland, Australia, Japan, Brazil, France, India, Ireland, Portugal, Sweden, Belgium
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a colorimetric assay used to measure cell metabolic activity. It is a tetrazolium-based compound that is reduced by living cells, producing a purple formazan product that can be quantified spectrophotometrically.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (373 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (292 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (118 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (102 mentions)
Streptomycin, by Thermo Fisher Scientific (92 mentions)
Trypsin edta, by Thermo Fisher Scientific (90 mentions)
Penicillin, by Thermo Fisher Scientific (82 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (72 mentions)
Propidium iodide, by Merck Group (69 mentions)
Rpmi 1640, by Thermo Fisher Scientific (51 mentions)
β actin, by Cell Signaling Technology (48 mentions)
Trypsin, by Thermo Fisher Scientific (47 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (46 mentions)
Fetal bovine serum (fbs), by Merck Group (46 mentions)
Methanol, by Merck Group (43 mentions)
Bovine serum albumin (bsa), by Merck Group (41 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (40 mentions)
Streptomycin, by Merck Group (36 mentions)
Hoechst 33342, by Merck Group (32 mentions)
Triton x 100, by Merck Group (32 mentions)
1 469 protocols using 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt
1
Cytotoxicity evaluation of FTY720 and compound 10
2
Evaluating SNX-2112 Cytotoxicity and Signaling
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SNX-2112 was synthesized as previously described in our lab with >98.0% purity [42 (link)], dissolved in Dimethyl sulfoxide (DMSO) to obtain a 100 mM stock solution, and stored at −20°C. TRAIL was purchased from Merck Millipore (Waltham, MA, USA). N-Acetyl-cysteine (NAC) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Bafilomycin A1 (BFA) was purchased from Selleck Chemicals (Shanghai, China). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Antibodies for GAPDH, Bcl-2, Bcl-xL, FLIP, pro-caspase 3, cleaved-caspase 3 (c-caspase 3), cleaved-caspase 8 (c-caspase 8), cleaved-PARP (c-PARP), Akt, p-Akt (Ser473), DR4, DR5, LC3, Beclin1, Atg7, p62, p-mTOR, p-S6, p-4EBP1, p53, p-ERK, ERK, p-p38, p38, p-JNK, and JNK were purchased from Cell Signaling Technology (CST; Beverly, MA, USA).
3
Synthesis and Characterization of Folic Acid-Conjugated Magnetic Nanoparticles
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All reagents and solvents were purchased commercially and used without further purification. Bovine serum albumin (BSA, fraction V, pH 7.0), folic acid (FA), NIR797 isothiocyanate, N-hydroxysuccinimide (NHS), N-dicyclohexylcarbodiimide (DCC), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Superparamagnetic Fe3O4 nanoparticles were prepared via the coprecipitation method as described previously [27] (link), [28] (link). Isoflurane was purchased from Shandong Keyuan Pharmaceutical Co., Ltd. (China).
4
Synthesis and Characterization of Novel Compounds
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2,4-dihydroxyacetophenone, 1-bromooctane, potassium carbonate (K2CO3), potassium iodide (KI), magnesium sulfate (MgSO4), potassium hydroxide (KOH), 3-bromobenzaldehyde, glutathione (GSH), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were all obtained from Sigma-Aldrich. Tetrahydrofuran (THF) was received from Tokyo Chemical Industry Co. Ltd. Dichloromethane (CH2Cl2), chloroform (CHCl3), and hexane were all purchased from Anaqua Global International Inc. Limited. SYTO 9 and propidium iodide (PI) were received from Thermal Fisher. Ethanol (EtOH) and methanol were obtained from Merk. Bovine serum albumin (BSA) was purchased from Aladdin. All commercial reagents were used as received unless otherwise stated. Ultrapure water (18.25 MΩ·cm, 25 °C) was used in the experiments.
5
Antioxidant and Anti-inflammatory Assays
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Folin-Ciocalteau reagent, 4-hydroxycinnamic acid (4-HCA), catechin, quercetin (QR), Ursolic acid (UA), butylated hydroxytoluene (BHT), 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS), α, α-diphenyl-β-picrylhydrazyl (DPPH), CCl4, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) probe, Ethidium bromide (EB), acridine orange (AO), thiobarbituric acid (TBA), reduced glutathione (GSH), propidium iodide (PI), 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT), and tetramethoxypropane (TMP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Ks drug was manufactured by Fresenius Kabi Company (Hamburg, Germany) where one film-coated tablet contains α-keto analogue of isoleucine, leucine, phenylalanine, valine, methionine with other amino acids including L-lysine, L-threonine, L-tryptophan, L-histidine, and L-tyrosine. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and trypsin were obtained from Lonza, USA. Gene JET RNA purification kit, cDNA synthesis kit, and 2X SYBR green master mix kit were supplied from ThermoScientific, USA. Primers for nuclear factor-kappa (NF-к) B and inducible nitric oxide synthase (iNOS) were purchased from Bioneer, Korea. Other chemicals were obtained with a high grade.
6
Lipid and Chemical Procurement Protocol
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Chemicals, of at least analytical grade, including lipoic acid, N-acetyl cysteine (NAC), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red, were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) or Fisher Scientific (Loughborough, UK) unless stated otherwise. Phospholipid containing docosahexaenoate (DHA) acyl chains, 1,2-di-(4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoyl)-sn-glycero-3-phosphocholine (Di22:6PC) was from Avanti Polar Lipids (Alabaster, AL, USA). Chromatography-grade organic solvents were from Fisher, Merck or VWR International, and were used as supplied.
7
Rat Primary Astrocyte Culture Protocol
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Hanks’ balanced salt solution (HBSS) Ca2+ and Mg2+ free ((Gibco, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), HBSS with Ca2+ and Mg2+ ((Gibco, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), papain (Sigma-Aldrich, St. Louis, MO, USA), DNase (Sigma-Aldrich, St. Louis, MO, USA), DMEM Low glucose (1 g/L) (PanEco, Moscow, Russia), DMEM High glucose (4 g/L) (PanEco, Moscow, Russia), antibiotic-antimycotic ((Gibco, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (Invitrogen, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), trypsin (Gibco, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA), Lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA), Griess reagent (Sigma-Aldrich, St. Louis, MO, USA), RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany), Quantikine Rat Il-6, R&D Systems, USA, 4-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich, St. Louis, MO, USA), Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA).
8
Formulating Targeted Sonosensitive Drug Delivery
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1,2-Dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPC) and 1,[2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine]-N-[amino(polyethylene glycol)] (DPPE-PEG2000-NH2) were purchased from Ponsure Biotechnology (Shanghai, China, Lot). The commercial SonoVue®, diluting with 5 mL of saline to form MBs, was purchased from Bracco Diagnostics Inc (Geneva, Switzerland). PTX was purchased from Dalian Meilun Biology Technology Co., Ltd (Dalian, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich Co., Ltd (Shanghai, China). RGD peptide was purchased from CornerStone Therapeutics (Shanghai), Ltd (Shanghai, China). 1-(3-Dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (EDC) and cholesterol were purchased from Sinopharm Chemical Reagent Co., Ltd (China).
MDA-MB-231 triple-negative breast cancer cell line was provided by the State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute (Shanghai, China). BALB/c nu/nu female mice, weighing 18–20 g, were provided by the Animal Experiment Centre of Shanghai Cancer Institute. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Shanghai Jiao Tong University and approved by the Animal Ethics Committee of Shanghai Cancer Institute (License no. SYXK (Hu) 2012-0001).
MDA-MB-231 triple-negative breast cancer cell line was provided by the State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute (Shanghai, China). BALB/c nu/nu female mice, weighing 18–20 g, were provided by the Animal Experiment Centre of Shanghai Cancer Institute. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Shanghai Jiao Tong University and approved by the Animal Ethics Committee of Shanghai Cancer Institute (License no. SYXK (Hu) 2012-0001).
9
Podophyllotoxin Synthesis and Cell Assays
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Podophyllotoxin, as starting material, was obtained from resin of rhizome of Podophyllum peltatum as previous described [16 (link)]. Further transformation into podophyllic aldehyde was conducted following the synthetic procedure described previously by us [15 (link)]. Etoposide was purchased from Sigma-Aldrich (#E1383, Saint Louis, MO, USA). Compounds were dissolved in DMSO (10−2 M) and further dilutions were performed in PBS Na+/K+. Final concentration of DMSO in assays were <0.001%.
Dimethyl sulfoxide (DMSO) was obtained from Merck (Darmstadt, Germany). Bovine serum albumin (BSA), trypan blue, urea, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 96-well plates and BCA protein assay kit were purchased from Thermo Scientific (Rockford, IL, USA). Heat inactivated fetal bovine serum (FBS), L-glutamine, penicillin–streptomycin (P-S), and 0.25% trypsin-EDTA were purchased from Gibco® (Gran Island, NY, USA), and 6-well, clear, flat-bottom plates were purchased from Corning (Corning, NY, USA). Cycloscope™ reagent for cell cycle analysis was purchased from Cytognos (Salamanca, Spain).
Dimethyl sulfoxide (DMSO) was obtained from Merck (Darmstadt, Germany). Bovine serum albumin (BSA), trypan blue, urea, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 96-well plates and BCA protein assay kit were purchased from Thermo Scientific (Rockford, IL, USA). Heat inactivated fetal bovine serum (FBS), L-glutamine, penicillin–streptomycin (P-S), and 0.25% trypsin-EDTA were purchased from Gibco® (Gran Island, NY, USA), and 6-well, clear, flat-bottom plates were purchased from Corning (Corning, NY, USA). Cycloscope™ reagent for cell cycle analysis was purchased from Cytognos (Salamanca, Spain).
10
Chickpea Seed Polysaccharide Extraction and Bioactivity
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Chickpea seeds were obtained from the National Gene Bank (Beijing, China). DEAE Sepharose Fast Flow was obtained from GE Healthcare Bio-Sciences Co. (Piscataway, NJ, USA). Dextran with different molecular weight (5,000–670,000 Da), griess reagent, arabinose (Ara), rhamnose (Rha), mannose (Man), galactose (Gal), glucose (Glc), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethyl-benzothiazolin-6-sulfonic acid) diammonium salt (ABTS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MS, USA). RPMI 1640 media, lipopolysaccharide (LPS), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were obtained from Gibco BRL Life Technologies (Thermo Fisher Scientific, NY, USA). Raw murine macrophage 264.7 (RAW 264.7) cells were purchased from the Cell Resources Center of the Chinese Academy of Sciences (Shanghai, China). OptEIA ELISA kits for tumor necrosis factor-α (TNF-α), MCP-1 and interleukin-6 (IL-6) were purchased from BD Biosciences (San Diego, CA, USA). A PathScan Antibody Array Kit was purchased from Cell Signaling Technology (Shanghai, China). All other chemicals and solvents used were analytical grade, unless otherwise specified.
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