Lipopolysaccharides from Escherichia coli O111:B4 (LPS; product #: L2630), Polyinosinic–polycytidylic acid sodium salt (Poly I:C; product #: P1530), thiazolyl blue tetrazolium bromide (MTT; product #: M5655), gentamycin (product #: G1397), curcumin (product #: C7727), 1α,25-dihydroxyvitamin D3 (product #: D1530), and progesterone (product #: PHR1142) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tri Reagent solution was obtained from the Molecular Research Centre (Cincinnati, OH, USA). All other chemicals were of analytical grade.
Thiazolyl blue tetrazolium bromide mtt
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Italy, China, Macao, Sao Tome and Principe, France
Thiazolyl blue tetrazolium bromide (MTT) is a yellow tetrazolium salt that can be reduced to a purple formazan product by dehydrogenase enzymes, primarily those found in the mitochondria of living cells. This reduction process is used as a widely accepted method for assessing cell viability and proliferation in various cell-based assays.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (43 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (39 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (27 mentions)
Propidium iodide, by Merck Group (13 mentions)
Fetal bovine serum (fbs), by Merck Group (7 mentions)
Phosphate buffered saline (pbs), by Merck Group (7 mentions)
Cisplatin, by Merck Group (6 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (6 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (6 mentions)
Sodium hydroxide (naoh), by Merck Group (6 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Trypsin edta, by Thermo Fisher Scientific (6 mentions)
Resazurin, by Merck Group (5 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Triton x 100, by Merck Group (5 mentions)
Rpmi 1640 medium, by Merck Group (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (5 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (5 mentions)
231 protocols using thiazolyl blue tetrazolium bromide mtt
1
Evaluating Immune Response Modulators
2
Splenocyte Viability Assay for BTWT Components
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SplenocyteS isolated from the spleen of mice were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml each of penicillin, and streptomycin (Gibico, Invitrogen, Carlsbad, CA, USA) overnight. To evaluate the cell viability, 2 × 105 splenocytes cultured in 96-well plates were treated with 1 µM of 11 components of BTWT for 24 h and then submitted to cell viability assay by mitochondria-dependent reduction of thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich). All experiments were repeated in quadruplicate in different intervals.
3
Synthesis and Characterization of Multifunctional Hydrogels
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All chemicals used were reagent-grade and used as purchased without further purification. Fmoc–Lys–Fmoc–OH amino acid (C36H34N2O6, Mw = 590.66 g/mol), 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane (U) (purity 98%), itaconic anhydride (ITA) (purity 98%), and 2,20-Azobis (2-methylpropionitrile) (AIBN) (purity 98%) were purchased from Sigma-Aldrich (Darmstadt, Germany). Fmoc-Gly-Gly-Gly-OH tripeptide (C21H21N3O6, Mw = 411.41 g/mol) was obtained from Bachem (Bubendorf, Switzerland). Alginic acid sodium salt from brown algae was supplied by Acros Organics (Geel, Belgium). Sodium phosphate buffer (PBS, pH 7.4, 0.01 M) was prepared using monosodium phosphate (NaH2PO4 × 2H2O) and disodium phosphate (Na2H2PO4 × 7H2O) via standard protocol. Dimethyl sulfoxide (DMSO) was acquired from Fluka (Buchs, Switzerland). Dulbecco’s Modified Eagles Medium (DMEM) high glucose (4500 mg/L glucose with L-glutamate and pyruvate), fetal bovine serum (FBS), antibiotics mixture (penicilin-streptomycin-neomycin mixture-P/S/N ≈5000 units penicilin, 5mg streptomycin, 10 mg neomycin/mL), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Steinheim, Germany), while the nutrient mixture HAM-F12 was purchased from Biological Industries, Beit-Haemek, Israel. The water used in the experiments was purified using an Ultra Clear TWF UV System.
4
Cell Viability Assay with MTT
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Cells were plated onto 96-well plates at seeding densities of 6.5 × 103 cells per well for PA-1, H1299 and SiHa cells and 7 × 103 cells per well for D54MG cells. The cell viability after treatment with appropriate agents was measured using Thiazolyl Blue Tetrazolium Bromide (MTT, Sigma) as previously described [17 (link)]. Concentrations of DHA that produced 50% inhibition in cell survival (IC50) following a 24 h exposure, were manually derived from dose–response curves generated by the Microsoft Excel 2010 edition.
5
Preparation and Characterization of Lipid Formulations
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Hydrogenated palm oil (Condea Chemie, Hamburg, Germany) was a gift from the Malaysian Palm Oil Board (MPOB) and olive oil was purchased from Basso Fegele and Figli Srl (San Michele di Serino, Italy). Others ingredients included were polysorbate 80 (Thermo Fisher Scientific, Waltham, MA, USA) Ultrapurified water (Merck Millipore, Billerica, MA, USA) and Lipoid S100 (lecithin) (Lipoid GmbH, Ludwigshafen, Switzerland).
Thimerosal, sorbitol, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Tamoxifen free base, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), ribonuclease A (Rnase A), thiazolyl blue tetrazolium bromide (MTT), Harris’s haematoxylin and eosin (H&E), ketamine hydrochloride, xylazine hydrochloride, horse serum, epidermal growth factor (EGF), hydrocortisone and insulin were purchased from Sigma-Aldrich (St Loius, MO, USA). Recombinant human erythropoietin was purchased from Peprotech (Rocky Hill, NJ, USA), paraformaldehyde (Acros Organics, USA), normal saline (0.9% NaCl), and 10% buffered formalin and Triton X–100 from Thermo Fisher Scientific (United States).
Rat mammary gland tumor cell (LA7) and non-tumorigenic breast (MCF-10A) cells were purchased from the American Type and Culture Collection (ATCC, Manassas, VA, USA).
Thimerosal, sorbitol, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Tamoxifen free base, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), ribonuclease A (Rnase A), thiazolyl blue tetrazolium bromide (MTT), Harris’s haematoxylin and eosin (H&E), ketamine hydrochloride, xylazine hydrochloride, horse serum, epidermal growth factor (EGF), hydrocortisone and insulin were purchased from Sigma-Aldrich (St Loius, MO, USA). Recombinant human erythropoietin was purchased from Peprotech (Rocky Hill, NJ, USA), paraformaldehyde (Acros Organics, USA), normal saline (0.9% NaCl), and 10% buffered formalin and Triton X–100 from Thermo Fisher Scientific (United States).
Rat mammary gland tumor cell (LA7) and non-tumorigenic breast (MCF-10A) cells were purchased from the American Type and Culture Collection (ATCC, Manassas, VA, USA).
6
Cytotoxicity Evaluation of RC and ARC Extracts
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The cytotoxicity was measured by using an 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay and aliquot cells (RAW 264.7 cells) at a density of 105 cells/ml into 96‐well plates and incubated them for 20 hr. After incubation, the plates were treated with different concentrations of RC and ARC extracts and incubated for 22 hr with 5% CO2 at 37°C. Each well was added with 5 mg/ml thiazolyl blue tetrazolium bromide (MTT, Sigma‐Aldrich Co.). After incubation for 2 hr in CO2 incubator, the supernatant was removed after centrifugation at 4℃ at 1747 g for 10 min. Each well then had dimethyl sulfoxide added (Sigma‐Aldrich Co.), and the plates were identified by a microplate reader (Model 550, Bio‐Rad) at 540 nm.
7
Cytotoxicity Evaluation of PC12 Cells
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PC12 cell line, derived from a rat pheochromocytoma, was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 6% fetal bovine serum, 6% horse serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a water-saturated 7.5% CO2 incubator. Cultured PC12 cells in 96-well-plate (15,000 cells/well) were pre-treated with various concentrations (0.1, 0.3, 1, 3, 10, 30, 100 μM) for 48 h. Cell viability test was performed with the addition of thiazolyl blue tetrazolium bromide (MTT) (Sigma, USA) in PBS at a final concentration of 0.5 mg/mL for 1 h. After the solution was removed, the purple precipitate inside the cells was re-suspended in DMSO and then measured at 570 nm absorbance.
8
MTT Assay for Cell Viability
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Cell lines were cultured at a density of 10.000 cells/well in 96-well plates, with or without periodic addition of SAMe (Sigma-Aldrich) at a final concentration of 10 μg/mL. At different times of culture (3, 5, 7 and 10 days), 10 μl of Thiazolyl Blue Tetrazolium Bromide (MTT, from Sigma-Aldrich) was added per well to achieve a final concentration of 0.45 mg/ml and incubated by 4 hours at 37°C. The medium was removed and 100 μl of Dimethyl sulfoxide (Sigma-Aldrich) was added per well to dissolve formazan crystals for 10 min and absorbance was recorded at 570 nm in a Nanoquant platform (Tecam).
9
Investigating Cellular Pathways and Toxicity
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Fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Gibco (Grand Island, NY, USA). Penicillin-streptomycin and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Hyclone (Logan, UT, USA). Dextrose, sodium bicarbonate, 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bicinchoninic acid (BCA) kit and SuperSignalTM west Femto chemiluminescent substrate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against Smad2/3 and kidney injury molecule-1 (KIM-1) were purchased from Cell Signaling (Denver, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore (Temecula, CA, USA). Antibodies against AhR, hypoxia-inducible factor 1-alpha (HIF-1α), epithelial cadherin (E-cad), fibronectin (FN), and Bcl-2-associated X protein (Bax), caspase-3 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). AhR siRNA (5′-GUGACUUGUACAGCAUAAUTT-3′) was purchased from GenePharma (Shanghai, China), Smad2/3 siRNA (sc-37238) was purchased from Santacruz Biotechnology (Santacruz, CA, USA), and HIF-1α siRNA (SASI_HS02_00332063) was purchased Sigma-Aldrich (St. Louis, MO, USA).
10
VEGF-induced Cell Proliferation Assay
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Recombinant human VEGF, thiazolyl blue tetrazolium bromide (MTT), 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT), phenylmethylsulfonyl fluoride (PMSF), and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). RIPA buffer and chemiluminescent substrate were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Dimethyl sulfoxide, sodium fluoride (NaF), and bovine serum albumin (BSA) were from Wako Pure Chemical Industries (Osaka, Japan). Bradford reagent was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). MRBH, also called MGN-3 or BioBran, was provided by Daiwa Pharmaceutical Co., Ltd. (Tokyo, Japan).
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