Once established, fibroblast cultures were trypsinized and plated in 5 sets of triplicates, one set for control and the others for test concentrations, onto a 96-well micro titer plate (10,000 cells/well) with FGM containing 10% FBS. After overnight incubation or when it reached 70–80% confluence, the medium was replaced with PBS overnight for cell starving. The cells were treated with varying concentrations of lignocaine (0.5–2.0%). The control wells were maintained with PBS. After 2 h of incubation, MTT assay was performed to analyze the cellular viability using MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue, Sigma-Aldrich, USA). The viability of cells was assessed at 2 h. After treatment, the cells were rinsed with 1× PBS and incubated with 0.5 mg/mL MTT diluted in complete DMEM for 2 h at 37°C. After incubation, DMEM with MTT was removed, formazan crystals were solubilized in 100 µL dimethyl sulfoxide (DMSO), and absorbance was read at 570 nm using a 96-well ELISA plate reader (Power Wave HT Microplate Spectrophotometer). The optical density of the formazan product produced by metabolic activity of the cells is directly proportional to the number of live cells.
Thiazolyl blue
Manufactured by Merck Group
Sourced in United States, Germany
Thiazolyl blue is a colorimetric reagent commonly used in cell proliferation assays. It is a water-soluble tetrazolium salt that can be reduced by metabolically active cells, producing a colored formazan product. The intensity of the color formed is directly proportional to the number of viable cells, making it a useful tool for quantifying cell viability and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Multiskan ascent microplate reader, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Formic acid, by Carl Roth (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Neubauer chamber, by Merck Group (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Isopropanol, by Carl Roth (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dmem low glucose medium, by Merck Group (1 mentions)
Aβ42 peptide, by Merck Group (1 mentions)
7s nerve growth factor, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Horse serum, by HiMedia (1 mentions)
Zoledronic acid, by Merck Group (1 mentions)
Tumor necrosis factor (tnf), by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
35 protocols using thiazolyl blue
1
Evaluating Lignocaine's Cytotoxicity on Fibroblasts
2
MTT Assay for Cell Viability
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For the MTT assay, cells were seeded in 96-well plates. After 72 h of incubation with Se and Cu, 20 μl of 5 mg/ml thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich) was added to the media. After 3 h, media were discarded followed by a 10 min shaking step with 5% (v/v) formic acid (Carl Roth, Karlsruhe, Germany) in 100% isopropanol (Carl Roth) to dissolve the obtained formazan crystals. Absorption was measured at 550 nm with 690 nm as reference wavelength, using a microplate reader (Synergy H1, Biotek, Bad Friedrichshall, Germany). As an additional assay for cell viability, the cell number was determined using a hemocytometer (Neubauer chamber) and trypan blue (Sigma-Aldrich).
3
Fabrication and Characterization of Gold Nanoparticles
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Gold nanospheres 5 nm (product code # 752568), 50 nm (# 753645) and 100 nm (# 753688) as well as Aβ42 peptide were purchased from Sigma Aldrich, USA. The AuNSs were stored at 4 °C and used as supplied. Ni–NTA agarose was obtained from Qiagen. IPTG and PMSF were purchased from Sigma Aldrich, USA. Urea was purchased from Calbiochem, India. All other chemicals were of analytical grade and were procured from Merck, India. Horse Serum was bought from Himedia Laboratories. Fetal Bovine Serum (FBS), trypsin–EDTA, penicillin and sterptomycin were obtained from Gibco (Thermo Fisher Scientific). Thiazolyl Blue, tetrazolium bromide (MTT), DMEM low glucose medium and Nerve Growth Factor-7S were purchased from Sigma Aldrich.
4
Cellular Cytotoxicity Assay Protocol
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Glyoxal, pyrrolidine dithiocarbamate (PDTC), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue (MTT) and zoledronic acid (ZA) were purchased from Sigma-Aldrich. Cyclosporin A (CsA, Sandimmun® 50 mg/mL) was from Novartis Pharma SAS (Rueil-Malmaison, France). The cytokines TNF and IFNγ were purchased from R&D Systems (Lille, France).
5
Assessment of Cell Viability via MTT Assay
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Cells were cultured in RPMI 1640 (Gibco-BRL, Grand Island, NY, USA) without phenol red and treated for 24 h with chitin or Car/Thy at concentrations ranging from 50 to 1000 μg/mL. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue, Sigma-Aldrich, Munich, Germany, Cat. No. M5655) was added to wells containing cells at a final concentration of 0.5 mg/mL. Enzymatic conversion of MTT to formazan was detected by for colorimetric analysis at a wavelength of 570 nm (BioTek ELx808, BioTek Instruments, Inc. Winooski, VT, USA). All tests were done in triplicate. Cells treated with Triton-X100 (1%, Sigma-Aldrich, Munich, Germany, Cat. No. T8787) were used as positive controls.
6
Detailed Reagent Acquisition Protocol
7
Cell Viability Assay with H2O2 Exposure
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The cells were transfected with siRNA into 48-well plates and then maintained for 48 h at 37℃ in an incubator in a 5% CO2 atmosphere. Cells were exposed to H2O2 in glucose/serum free DMEM for 3 h and then further incubated for 6 h in the normal medium supplemented with glucose and serum. Thiazolyl blue (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenytetrazolium bromide reagent, Sigma) was added to each well, to a final concentration of 0.5 mg/ml. After incubation at 37℃ in an incubator under a 5% CO2 atmosphere, the solution was suctioned and 200 µl of acid isopropyl alcohol (Sigma) was added to each well. Absorption was then measured at a wavelength of 570 nm. After extraction with background value, the relative viability was determined by ratio of values to those of control in terms of mean±standard errors.
8
Diamide-Induced Cellular Reduction Assay
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0.3 mM diamide in RPMI 1640 was administered at distinctive times to the T cell samples in 96-well plate (Corning, NY, USA) in order to establish reduction activity. Next, 5 mg/mL Thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) was incubated for 4 h. After the incubation, the medium was replaced with 0.3 mL MTT solvent solution (4 mM HCl, 0.1% Nondet P-40 in isopropanol) in order to solubilize the converted dye. Microplate reader (Biorad, Hercules, CA, USA) was used to measure the absorbance at 570 nm. The reduction activity is showed as percentage of the compared control.
9
MTT Assay for Cell Viability
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In 96-well plates, 500 cells per well for DAOY cells and 5 × 103 cells per well for D283Med cells were seeded in sextuplicate. After 72 h of incubation at 37 °C, 0.5 mg/mL Thiazolyl Blue Tetrazolium Bromide (MTT, Sigma, St. Louis, MO, USA) was added, and after additional 3 h of incubation at 37 °C, the contents of the wells were removed, and formed crystals were resuspended in 150 μL DMSO per well for DAOY cells. However, for D283Med cells, 100 μL of isopropanol + 0.01 M HCl per well were added to the culture media, and the crystals were resuspended by pipetting up and down. In both cell lines, absorbance was measured at 570 nm in a MultiSkan Ascent microplate reader (Thermo Scientific, Budapest, Hungary) using the Ascent software. Cellular viability of the shERBB4 cells was calculated relative to the absorbance of control cells.
10
Investigating EGCG's Anti-Cancer Mechanisms
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EGCG and thiazolyl blue were purchased from Sigma (St. Louis, MO, USA). RPMI-1640 basal medium and fetal bovine serum (FBS) was produced by HyClone (Logan, UT, USA). RPMI-1640 basal medium and EGCG were used to prepare a 10 mmol/l storage solution. It was sterilized by filtration (0.22 µm) and stored at −80°C in the dark. MTT was dissolved in phosphate-buffered saline (PBS) solution at 5 mg/ml, and was stored at −20°C in the dark after sterilization. Mouse anti-human P53, Bcl-2 and GAPDH were all purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and the RT-PCR kit was purchased from Takara (Otsu, Shiga, Japan). P53, Bcl-2 and GAPDH primers were produced by Invitrogen (Carlsbad, CA, USA) (Table I ) and si-P53 was supplied by Gemma (Shanghai, China).
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