MCF-7 cells under normal conditions and MCF-7 LTED cells (27 weeks) were seeded (3 × 103 per well) in a 96-well plate. After 24 hours of incubation, tamoxifen was added at 1.25 μM, 2.5 μM, 5 μM, and 10 μM. Each treatment was carried out in triplicate including control untreated cells. The plates were then incubated for 24 hours, then were analyzed using the MTT assay. The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was purchased from Sigma-Aldrich. MTT solution was prepared at 5 μg/mL in PBS buffer; then, 20 μL of the MTT solution was added per well, and then, the plate was incubated at 37°C for 1 hour. Finally, the medium was removed, and 200 μL of DMSO was added to solubilize the formazan salt. The plate was analyzed using a microplate reader (Microplate Autoreader EL311, BioTek Instruments Inc., Winooski, Virginia, USA) at 570 nm to determine the optical density (OD).
Mtt reagent 3 4 5 dimethylthiazol 2 yl 2 5 diphenyl tetrazolium bromide
Manufactured by Merck Group
Sourced in United States
The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a chemical compound used in colorimetric assays. It is a yellow tetrazolium salt that is reduced to a purple formazan product by metabolically active cells, providing a quantitative measure of cell viability and proliferation.
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Lab products found in correlation
Microplate autoreader el311, by Agilent Technologies (1 mentions)
Chlorpromazine, by Merck Group (1 mentions)
Hank s balanced salt solution hbss, by Merck Group (1 mentions)
Propylene glycol, by Merck Group (1 mentions)
Tryple express enzyme no phenol red, by Thermo Fisher Scientific (1 mentions)
Kolliphor el, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Labrasol, by Gattefosse (1 mentions)
Lauroglycol fcc, by Gattefosse (1 mentions)
Transcutol hp, by Gattefosse (1 mentions)
Rpmi 2650, by American Type Culture Collection (1 mentions)
Enspire, by PerkinElmer (1 mentions)
Spectra id3 max, by Molecular Devices (1 mentions)
Hb 8065, by American Type Culture Collection (1 mentions)
Penicillin streptomycin mixture, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
5 protocols using mtt reagent 3 4 5 dimethylthiazol 2 yl 2 5 diphenyl tetrazolium bromide
1
MTT Assay for Tamoxifen Sensitivity
2
In Vitro Cytotoxicity Evaluation
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Chlorpromazine, Kolliphor EL, Miglyol 840, dimethyl sulfoxide, glycerol, propylene glycol, polyvinyl alcohol, and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MI, USA). Lauroglycol FCC, Transcutol HP, Labrafil 1944, and Labrasol were gifted by Gattefossé (Lyon, France). The human nasal epithelial cell line (RPMI 2650) was from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). The MTT reagent 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and the buffer solutions, such as Hank’s Balanced Salt Solution (HBSS) and phosphate-buffered saline (PBS), were purchased from Sigma-Aldrich (St. Louis, MI, USA). The RPMI cell culture maintenance medium solution, TrypLE™ Express Enzyme (no phenol red), was ordered from Thermo Fisher Scientific (Waltham, MA, USA). Ninety-six-well cell plates and culturing flasks were obtained from VWR International (Debrecen, Hungary).
3
Evaluating Microencapsulated Cell Functionality
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Several assays were conducted to assist in the assessment of the microencapsulated cells’ biological activity induced by glucose. Assays including glucose-induced cellular insulin release studies with an ultrasensitive mouse insulin ELISA kit (Mercodia Cooperation, Uppsala, Sweden) and MTT studies were used to assess glucose-induced viability. MTT reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical Co. St. Louis, MO, USA) was utilised in a validated method, previously established by the laboratory, allowing accurate assessment of cell viability in microcapsules without having to rupture the capsules [33 (link),34 (link)]. Measurements of antioxidant activity were taken using a plate reader (Enspire, PerkinElmer, Waltham, MA, USA) for fluorescence, with increased fluorescent readings indicating increased oxidative stress, and therefore, decreased antioxidant activity. Oxidative stress measurements were taken from oxidized radical species following incubation with a mixture of dichloro-dihydro-fluorescein diacetate and 2,2′-azobis-2-methyl-propanimidamide dihydrochloride [25 (link),37 (link),38 (link)]. Assessments were taken under glycaemic and hyperglycaemic conditions.
4
Hepatic Safety Evaluation of HBK-10
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To investigate the preliminary hepatic safety of HBK-10 at the concentration used in metabolic stability study, a human hepatocellular carcinoma cell line—HepG2 (HB-8065TM, ATCC, Manassas, VA, USA) was used in the study. HepG2 cells were cultured in standard conditions (37 °C, 5% CO2, 95% humidity) in appropriate culture medium (according to manufacturer procedure), supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics (1% streptomycin/penicillin mixture; Sigma-Aldrich, Steinheim, Germany). Cells were seeded in 96-well plates at density of 1 × 104 per well. After 24 h cells were treated with two doses of HBK-10 (5 and 25 µM, solvent—PBS) and incubated for additional 24 h. Following the incubation cells viability was measured with MTT assay. For this purpose, MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma Aldrich, Steinheim, Germany) was added to each well at the concentration 0.5 mg/mL. After 4 h incubation at 37 °C, the medium was aspirated, and formazan crystals produced in the cells were dissolved in DMSO. Then the absorbance of solution was determined at 570 nm (A570) on plate reader (Spectra iD3 Max, Molecular Devices; San Jose, CA, USA). Viability (% of control) was determined by dividing A570 of experimental wells by of A570 of control wells × 100%.
5
Preparation and Characterization of Mucoadhesive Hydrogel
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HEMA, ethylene glycol dimethacrylate (EGDMA), inhibitor remover, methacrylic acid (MAA), sodium dodecyl sulfate (SDS), benzoyl peroxide (BPO), DTT, potassium bromide (KBr), 1-butanol, dexamethasone, 3AAPBA, bovine submaxillary mucin (BSM), and MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) were purchased from Sigma-Aldrich. BAC was purchased from Alfa Aesar and potassium chloride (KCl) was purchased from EMD chemicals. Spectra/Por® 6 regenerated cellulose 50 kDa molecular weight cut off (MWCO) dialysis tubing was purchased from Spectrum® Laboratories. Acrodisc CR 13 mm high pressure liquid chromatography (HPLC) grade syringe filter with a 0.2 μm pore size were purchased from PALL Life Sciences.
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