Fresh herbs of Picria fel-terrae Lour. was collected from Tiga Lingga village, Dairi regency, Sumatera Utara province, Indonesia. Picria fel-terrae Lour. was identified in Research Centre for Biology, Indonesian Institute of Science, Bogor, and the voucher specimen was deposited in herbarium. Chemicals used were AlCl3.6H2O (Merck), annexin-V (BioLegend), distilled water, DMSO (Sigma), 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Sigma), [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) (Sigma), folin ciocalteu, propidium iodide kit (BioLegend), quercetin (Sigma), sodium acetate (Merck), sodium bicarbonate (Merck).
3 4 5 dimethylthiazole 2 yl 2 5 diphenyltetrazolium bromide mtt
Manufactured by Merck Group
Sourced in United States
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a chemical compound commonly used as a colorimetric assay for measuring the activity of enzymes that reduce metabolic substrates, thus indicating the general viability and metabolic activity of cells in cell culture. It provides a quantitative measure of viable cell number.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Anti actin, by Merck Group (2 mentions)
C 17 erbb 3, by Santa Cruz Biotechnology (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Single bond universal, by 3M (2 mentions)
Filtek z350 xt, by 3M (2 mentions)
Ethanol, by Merck Group (2 mentions)
Acetonitrile, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Annexin 5, by BioLegend (1 mentions)
Alcl3 6h2o, by Merck Group (1 mentions)
1 1 diphenyl 2 picrylhydrazyl dpph, by Merck Group (1 mentions)
Quercetin, by Merck Group (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)
24 protocols using 3 4 5 dimethylthiazole 2 yl 2 5 diphenyltetrazolium bromide mtt
1
Phytochemical and Biological Evaluation of Picria fel-terrae
2
ErbB-3 Signaling Pathway Antibody Protocol
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Antibodies used in the present study were as follows: phosphorylated ErbB-3 (Clone 21D3, Tyr1289, #4791), ErbB-3 (Clone D22C5, #12708), phosphorylated Akt (Ser473, Clone D9E, #4060), Akt (#9272), all from Cell Signaling Technology, Inc. Neuregulin-1β (NRG-1β, #5218SC) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). EV20 antibody was produced as previously described [13 (link)]. MP-RM-1 antibody was produced as previously described [20 (link)]. Recombinant human ECD HER-3 was from ACROBiosystems (Bethesda, MD, USA). (3-(4,5-Dimethylthiazole-2-yl)-2,5 diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).
3
Cytotoxicity Evaluation of Extracts
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Cytotoxicity of the extracts was evaluated by measuring the viability of Human Cardiac Microvascular Endothelial Cells (HCMEC) obtained from the American Type Culture Collection (ATCC). Cells were seeded in a 96-well plate with a final concentration of 10 × 104 cells mL−1 with Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich (St. Louis, MO, USA) for 24 h.
The cellular viability was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich (St. Louis, MO, USA) to formazan, quantified by optical density measurement at 510 nm, as described by Lopes et al. [74 (link)]. Several concentrations of the extracts were tested: 50, 100, 200, and 300 μg·mL−1—using DMSO 1% as negative control and DMSO 20% as the positive control. The assay was independently repeated four times, with duplicate extracts. Cytotoxicity was expressed as a percentage of cell viability, considering the values of the negative control as 100% viability.
The cellular viability was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich (St. Louis, MO, USA) to formazan, quantified by optical density measurement at 510 nm, as described by Lopes et al. [74 (link)]. Several concentrations of the extracts were tested: 50, 100, 200, and 300 μg·mL−1—using DMSO 1% as negative control and DMSO 20% as the positive control. The assay was independently repeated four times, with duplicate extracts. Cytotoxicity was expressed as a percentage of cell viability, considering the values of the negative control as 100% viability.
4
Signaling Pathways Activation Assay
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Antibodies were as follows: phosphorylated EGFR (Tyr1068), phosphorylated ErbB-2 (Tyr1248), phosphorylated ErbB-3 (Tyr1289), phosphorylated Akt (Ser473), phosphorylated ERK1/2, Akt and ERK1/2 from Cell Signaling Technology (Danvers, MA, USA); C-17 ErbB-3 and C-18 ErbB-4 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); ErbB-2 antibody for western blot was kindly provided by Dr O Segatto; anti-Actin was from Sigma-Aldrich Corporation (St. Louis, MO, USA). Herceptin (trastuzumab) was from Roche Pharma AG (Grenzach-Wyhlen, Germany), Erbitux (cetuximab) was from Merck KGaA (Darmstadt, Germany). NRG-1β was purchased from R&D (R&D Systems, Inc., Minneapolis, MN, USA) or Cell Signaling Technology. 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Corporation.
5
Antimicrobial Peptide Characterization
6
PLGA-PBAE Polyplexes for Luciferase Delivery
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PLGA 50 : 50 RESOMER (RG502 H) polymer was purchased from Evonik Röhm Pharma GmbH, Germany. PBAE polymers A1 (bis(3-(propionyloxy)propyl)3,3′-(propane-1,3-diyl-bis(methylazanediyl))dipropanoate) and A2 (bis(4-(propionyloxy)butyl)3,3′-(ethane-1,2-diyl-bis(isopropylazanediyl))dipropanoate) were synthesized in our lab. Plasmid encoding firefly luciferase (pCMV-Luc) was procured from ELIM Biopharmaceuticals, USA, and propagated in large quantity in competent E. coli DH5α cells. Further the plasmid was isolated and purified using mini preparation kit from Sigma chemical Co, (St. Louis, MO, USA). Bright glow luciferase assay system was procured from Promega, USA. Quant-iT PicoGreen dsDNA Reagent and Kits were procured from Invitrogen Life Technologies Co. (USA). 3-(4,5 dimethyl thiazole-2 yl)-2,5-diphenyl tetrazolium bromide (MTT) from Sigma chemical Co, St. Louis, MO, USA was used in studies. Dimethylsulfoxide (DMSO), isopropanol were procured from Qualigens fine chemicals, Mumbai, India. Trypsin, 4-(2-hydroxyethyl)-1-Piperazineethanesulphonic acid (HEPES) sodium salt buffer saline, and normal melting agarose were procured from Himedia lab Pvt. Ltd., Mumbai, India. Lipofectamine 2000 and fetal bovine serum (FBS) were purchased from life technologies, Carlsbad, USA. All other chemicals used in the study were of analytical grade.
7
Fmoc-Phe-Phe-OH Hydrogel Cytotoxicity Analysis
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Lyophilized Fmoc-Phe-Phe-OH (FmocFF) was purchased from Bachem (Budendorf, Switzerland). Sodium alginate, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), fluorescein diacetate, propidium iodide, 4-Methylumbelliferyl phosphate (4-MUP), Rhodamin B, and Alizarin red were purchased from Sigma Aldrich (Rehovot, Israel). MC3T3-E1 preosteoblast cells were purchased from ATCC (Manassas, VA, USA). Pure Link RNA Mini Kit was purchased from Invitrogen; Thermo Fisher Scientific (Carlsbad, CA, USA), qScript cDNA Synthesis Kit and SYBR green reagents with Perfecta SYBR Green FastMix reagent with specific gene primers were purchased from Quanta Biosciences (Boston, BA, USA).
8
Cytotoxicity Evaluation of Cancer Cell Lines
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Human breast carcinoma (MCF-7) and colon cancer (HT-29) cell lines were purchased from Pasteur Institute of Iran (Tehran, I.R. Iran). Roswell park memorial institute (RPMI) medium 1640, fetal bovine serum (FBS), streptomycin and penicillin were provided from Gibco Invitrogen Corporation (UK). Pipettes, tissue culture flasks, 96-well plates, trypan-blue, and 3-[4,5-dimethylthiazole-2-yl]- 2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich Co. (UK). Dimethyl sulfoxide (DMSO) was from Merck (Darmstadt, Germany). UV/visible spectrophotometer (Shimadzu, 2100; Japan).
9
Antimicrobial Activity Evaluation
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Amikacin disulfate salt and streptomycin sulfate (Acros Organics) and kanamycin sulfate (Thermo Scientific), as well as thymol and piperine, were purchased from Sigma-Aldrich. Dimethyl sulfoxide (DMSO), p-iodonitrotetrazolium chloride (INT), and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were also purchased from Sigma-Aldrich. SSA and MHB media were purchased from Dominique Dutscher SAS, France.
10
ErbB-3 Signaling Pathway Analysis
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Antibodies were as follows: phosphorylated Akt (Ser473/Thr308), Akt, phosphorylated ErbB-3 (Tyr1289), ErbB-3, from Cell Signaling Technology (Danvers, MA); C-17 ErbB-3 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-actin was purchased from Sigma-Aldrich Corporation (St Louis, MO). Neuregulin-1β (NRG-1β) was purchased from R&D Systems, Inc (Minneapolis, MN). Recombinant human ECD ErbB-3 was from ACROBiosystems (Bethesda, MD). (3-(4,5-Dimethylthiazole-2-yl)-2,5 diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Corporation. EV20 antibody was produced as described [12 (link)].
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