Briefly, Vero cells were seeded into 96-well cell culture microplates and incubated overnight at 37 °C with 5% CO2. After the incubation, the cell monolayers were treated with different concentrations of Streptomyces KSF 103 ME up to 500 µg/mL in duplicate. The treated cells were incubated for four days at 37 °C with 5% CO2. After the incubation, MTS reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was added to each well, and the microplate was incubated at 37 °C for 4 h. To determine the cell viability, the optical density (OD) of the treated cells was measured at 490 nm to 700 nm using TECAN 96-well plate reader (TECAN, Männedorf, Switzerland). Dose–response curves were plotted using Graph Pad Prism Version 5 (Graph Pad Software Inc., San Diego, CA, USA), and the values of 50% cytotoxic concentration (CC50) and Maximum Non-Toxic Dose (MNTD) were calculated.
3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts reagent
Manufactured by Promega
Sourced in United States, United Kingdom
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) is a colorimetric assay reagent used for quantifying cell viability and proliferation. The MTS reagent is reduced by metabolically active cells, producing a colored formazan product that can be measured spectrophotometrically.
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Lab products found in correlation
Cresyl violet acetate, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Penicillin streptomycin solution, by Merck Group (2 mentions)
2 7 dichlorofluorescin diacetate dcfda, by Merck Group (2 mentions)
Zncl2, by Thermo Fisher Scientific (2 mentions)
Monochlorobimane mcb, by Thermo Fisher Scientific (2 mentions)
Cuso4 5h2o, by Thermo Fisher Scientific (2 mentions)
Phalloidin cruzfluor 647 conjugate, by Santa Cruz Biotechnology (2 mentions)
Dichloromethane dcm, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
96 well plate reader, by Tecan (1 mentions)
Prism version 5, by GraphPad (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Roche (1 mentions)
Nonidet p 40, by AppliChem (1 mentions)
Rnase a and pi, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
10 protocols using 3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts reagent
1
Cytotoxicity Evaluation of Streptomyces KSF 103 ME
2
Comparative Evaluation of NaB, Cur, and Pip
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NaB was purchased from National Boron Research Institute-BOREN (Ankara, Turkey). NaB stocks were prepared as 10 mg/mL in completed media using vortex and all solutions were filtered with a 0.22 μm filter before use. Cur and Pip were purchased from Sigma-Aldrich (St. Louis, MO, USA) and resuspended in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO) to prepare a stock solution of 50 mM and 150 mM, respectively. The main stock solution was filtered through a 0.22 μm filter and kept at − 20 °C for long time storage.
MTS reagent ([3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega (Southampton, UK). Annexin V-FITC Apoptosis Detection Kit was purchased from Roche (Germany). RNase A and PI were obtained from Sigma-Aldrich (Germany). Nonidet P-40 was obtained from AppliChem (Germany). RNeasy Mini kit, Sensiscript Reverse Transcription PCR Kit and QuantiTect SYBR Green PCR kit were supplied by Qiagen (United States).
MTS reagent ([3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega (Southampton, UK). Annexin V-FITC Apoptosis Detection Kit was purchased from Roche (Germany). RNase A and PI were obtained from Sigma-Aldrich (Germany). Nonidet P-40 was obtained from AppliChem (Germany). RNeasy Mini kit, Sensiscript Reverse Transcription PCR Kit and QuantiTect SYBR Green PCR kit were supplied by Qiagen (United States).
3
Cytotoxicity Assay and Apoptosis Analysis
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Phosphate buffered saline (PBS), L-glutamine, penicillin-streptomycin solution (10,000 U/mL, 10 µg/mL), and trypsin were from Sigma-Aldrich (St. Louis, MO, USA). MTS reagent 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium was purchased from Promega (Madison, WI, USA). Low- and normal-melt agarose were purchased from Helicon (Moscow, Russia). Basal Medium Eagle (BME), McCoy’s 5A Modified Medium (McCoy’s 5A), Roswell Park Memorial Institute medium (RPMI-1640), fetal bovine serum (FBS), agar, gentamicin, and Pageruler Plus Prestained Protein Ladder were purchased from ThermoFisher Scientific (Waltham, Massachusetts, USA).
Cell lysis buffer (10X); primary antibodies against caspase 9, caspase 3, cleaved caspase 3, Bcl-XL, Bax, and β-actin; and horseradish peroxidase (HRP)-conjugated secondary antibody from rabbit and mouse were obtained from Cell Signaling Technology (Danvers, MA, USA).
Cell lysis buffer (10X); primary antibodies against caspase 9, caspase 3, cleaved caspase 3, Bcl-XL, Bax, and β-actin; and horseradish peroxidase (HRP)-conjugated secondary antibody from rabbit and mouse were obtained from Cell Signaling Technology (Danvers, MA, USA).
4
Cell Viability and Oxidative Stress Assays
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Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute medium (RPMI 1640), Minimum Essential Medium (MEM), 1% penicillin-streptomycin solution and 10% fetal bovine serum (FBS) were purchased from Biolot (Moscow or St. Petersburg, Russia), penicillin-streptomycin solution, 10 μg/mL, and dimethyl sulfoxide (DMSO) from Sigma-Aldrich, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) from Promega (Madison, WI, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Sigma-Aldrich (St. Louis, MO, USA), 2,7-dichlorodihydrofluorescein diacetate solution (10.0 µM H2DCFDA) from Molecular Probes (Eugene, OR, USA), and cisplatin from VeroPharm (Moscow, Russia).
5
Cytotoxicity and Oxidative Stress Assays in Skin Cells
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Bovine skin gelatin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reagent), 4′,6-diamidino-2-phenylindole (DAPI), phenazine methosulfate (PMS), and Triton X-100 and 2′,7′-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma-Aldrich. Monochlorobimane (MCB) was purchased from ThermoFisher Scientific. CuSO4·5H2O, ZnCl2, CoCl2·6H2O, glutaric dialdehyde (GDA), and cresyl violet acetate were obtained from Acros Organics. Citrus pectin (classic CM 201) was obtained from Herbstreith&Fox.
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) was purchased from Promega. Phalloidin CruzFluor™ 647 conjugate, anti-VEGF (C-1) mouse monoclonal, anti-ICAM-2 (S-16) goat polyclonal, anti-MMP-3 goat monoclonal and anti MMP-3 goat monoclonal antibodies were purchased from Santa Cruz Biotechnology. Anti-HIF-1a mouse monoclonal antibody, donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647, and donkey anti-goat IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 555, were obtained from ThermoFisher Scientific. Anti-CD31 (PECAM-1) rabbit monoclonal antibody was obtained from Abcam. Hematoxylin and Eosin, and Giemsa staining were purchased from BioVitrum (Russia). Cell culture media and reagents were purchased from Paneco (Russia).
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) was purchased from Promega. Phalloidin CruzFluor™ 647 conjugate, anti-VEGF (C-1) mouse monoclonal, anti-ICAM-2 (S-16) goat polyclonal, anti-MMP-3 goat monoclonal and anti MMP-3 goat monoclonal antibodies were purchased from Santa Cruz Biotechnology. Anti-HIF-1a mouse monoclonal antibody, donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647, and donkey anti-goat IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 555, were obtained from ThermoFisher Scientific. Anti-CD31 (PECAM-1) rabbit monoclonal antibody was obtained from Abcam. Hematoxylin and Eosin, and Giemsa staining were purchased from BioVitrum (Russia). Cell culture media and reagents were purchased from Paneco (Russia).
6
Peptide Synthesis and Cell Culture Assays
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9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, fatty acids, Rink amide MBHA resin, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from NovaBiochem. Other chemicals including dichloromethane (DCM), dimethylformamide (DMF), acetonitrile, piperidine, N,N-diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), and triisopropylsilane (TIPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4′,6-diamidino-2-phenylindole (DAPI), phenazine methosulfate (PMS), Triton X-100, and 2′,7′-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Monochlorobimane (MCB) was purchased from ThermoFisher Scientific (Waltham, MA, USA). CuSO4·5H2O, ZnCl2, MnSO4·5H2O, and cresyl violet acetate were obtained from Acros Organics (Geel, Belgium).
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) was purchased from Promega (Madison, WI, USA). Phalloidin CruzFluor™ 647 conjugate was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). PC-12 and NIH 3T3 cell lines were obtained from the American Type Culture Collection (Manassas, VG, USA). Cell culture media and reagents were purchased from Paneco (Moscow, Russia).
4′,6-diamidino-2-phenylindole (DAPI), phenazine methosulfate (PMS), Triton X-100, and 2′,7′-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Monochlorobimane (MCB) was purchased from ThermoFisher Scientific (Waltham, MA, USA). CuSO4·5H2O, ZnCl2, MnSO4·5H2O, and cresyl violet acetate were obtained from Acros Organics (Geel, Belgium).
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) was purchased from Promega (Madison, WI, USA). Phalloidin CruzFluor™ 647 conjugate was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). PC-12 and NIH 3T3 cell lines were obtained from the American Type Culture Collection (Manassas, VG, USA). Cell culture media and reagents were purchased from Paneco (Moscow, Russia).
7
Adipogenic Differentiation Pathway Analysis
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Oil red O, Dex, insulin, IBMX, and SB203580 (a p38 inhibitor, a positive control) were purchased from Sigma-Aldrich Chemical Co.. Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were purchased from HiMedia Laboratories Pvt. Ltd.. Roswell Park Memorial Institute (RPMI) 1640 was purchased from Welgene. Penicillin-streptomycin and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphen-yl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent from Promega Corporation. Adiponectin, fatty acid-binding protein 4 (FABP4), PPARγ, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology, Inc. and horseradish peroxidase (HRP)-conjugated secondary antibodies from ABclonal Technology.
8
PEG-based Neuronal Cell Culture Assay
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Fumaryl chloride (95%), triethylamine chloride (≥99%), poly(ethylene glycol) (PEG) (1500 Da), poly(ethylene glycol) diacrylate (PEGDA, Mn = 575), N,N,N′,N′- tetramethylethylenediamine (TEMED, ≥99%), ammonium persulfate (APS), and dichloromethane (DCM) were purchased from Sigma-Aldrich (Saint Louis, MO, USA).
Cell culture media and reagents were purchased from Paneco (Moscow, Russia). The 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) was purchased from Promega (Madison, WI, USA). PC-12 rat pheochromacytoma and SH-SY5Y human neuroblastoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Cresyl violet acetate (Acros Organic) and 4,6-diamino-2-phenylindol (DAPI) (Sigma-Aldrich) were used for cell staining.
Cell culture media and reagents were purchased from Paneco (Moscow, Russia). The 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) was purchased from Promega (Madison, WI, USA). PC-12 rat pheochromacytoma and SH-SY5Y human neuroblastoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Cresyl violet acetate (Acros Organic) and 4,6-diamino-2-phenylindol (DAPI) (Sigma-Aldrich) were used for cell staining.
9
Cytotoxicity assessment of novel compounds
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HEK293 (0.8 × 103/200 µL), SK-MEL-28 (0.8 × 103/200 µL), HuTu80 (1.0 × 103/200 µL), and ZR-75-1 (1.2 × 103/200 µL) cells were seeded into 96-well plates (Jet Biofil, Guangzhou, China) for 24 h at 37 °C in a 5% CO2 incubator. Then cell monolayer was treated either with DMSO (control), Doxo (positive control) (5, 25, 50, 100 µM) or various concentrations of compounds 1 , 4 , and 5 and the mixture of 2 and 3 (10, 50, 100 µM) in fresh appropriate culture medium for 24 h. Subsequently, the cells were incubated with 15 µL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS reagent) (Promega, Madison, WI, USA) for 3 h, and the absorbance of each well was measured at 490/630 nm using Power Wave XS microplate reader (BioTek, Wynusky, VT, USA). The concentration at which the compounds exert half of its maximal inhibitory effect on cell viability (IC50) was calculated by the AAT-Bioquest® online calculator [31 ].
10
Chitosan-Chondroitin Sulfate Scaffold Characterization
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Low molecular weight (116 kDa) chitosan (CH) (Ref: 448869), chondroitin sulfate (CS) (C9819), phosphate buffered saline tablets (PBS), Dulbecco's modified Eagle's medium (DMEM) low glucose, Dulbecco's modified Eagle's medium without phenol red, sodium bicarbonate and dexamethasone were purchased from Sigma Aldrich (Germany). Fetal bovine serum (FBS) was purchased from Biochrom AG (Germany) and antibiotic-antimycotic (A/B) solution from Gibco (Spain). (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) MTS reagent was purchased from Promega (G5421, UK). Chitosan was further purified by the precipitation procedure, all the other chemicals were used with no further purification. After chitosan purification, the final deacetylation degree was 75-85%. ELISA kits for the detection of human PDGF-BB and TGF-b1 were purchased from R&D Systems.
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