To determine cell viability, assay was carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltretrazolium bromide (MTT, Thiazolyl blue, Carlroth, #4022.2) colorimetric assay, based on the reduction of tetrazolium salt. The HT22 cells were inoculated in a 96-well (1 × 105 cells/well) and treated with conditioned media for 24 h. After incubation, 10 μl of the MTT working solution (5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. After removing the media, formazans into the cells were dissolved with 150 μl dimethyl sulfoxide (DMSO). Absorbance at 540 nm was measured using a microplate reader (OpsysMR, DYNEX technologies, #1MRA-2067), and cell viability was determined as the percentage of MTT reduction, assuming the absorbance of control cells as 100%.
Thiazolyl blue
Manufactured by Carl Roth
Sourced in Germany
Thiazolyl blue is a tetrazolium-based compound commonly used in colorimetric assays for cell viability and proliferation. It serves as a redox indicator, undergoing reduction by cellular dehydrogenases to generate a colored formazan product that can be quantified spectrophotometrically. The core function of thiazolyl blue is to provide a measurable readout of metabolic activity in cell-based experiments.
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Lab products found in correlation
Infinite 200 pro reader, by Tecan (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Triton x 100, by Carl Roth (1 mentions)
0.1 m hcl, by Thermo Fisher Scientific (1 mentions)
Centro lb 960 luminometer, by Berthold Technologies (1 mentions)
Cell proliferation elisa kit, by Roche (1 mentions)
Multiskan fc photometer, by Thermo Fisher Scientific (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
Victor3, by PerkinElmer (1 mentions)
8 protocols using thiazolyl blue
1
MTT Assay for Cell Viability
2
Cell Proliferation Assay Using MTT
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To determine proliferation rates, cells were seeded on 6-well plates at a density of 1 × 105 cells per well. The cell viability was determined 1, 3 and 6 days after seeding using MTT assay by adding 90 μl of 5 mg/ml Thiazolyl blue ≥98% (Carl Roth; 4022) to each well. After 1h, the medium was aspirated, and stained cells were dissolved in 400 μl lysis buffer (80% isopropanol, 10% 1 M HCl, 10% Triton X-100) and diluted further if necessary. Absorption was measured at 595 nm using a plate reader. All values were normalized to day 1 to compensate for variations in seeding density. The mean value of three biological replicates was determined.
3
Cell Viability Assay for TNBC
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50 μl of indicated TNBC cell lines were added into each well of a transparent 384-well plate at density 1,500 cells/well. The cells were maintained in DMEM containing 10% FBS and incubated at 37°C, 5% CO2 overnight. The next day, the medium of each well was replaced by 50 μl fresh medium containing indicated concentrations of compounds. After incubation for 3–4 days, the medium in each well was replaced by 50 μl of 0.5 mg/ml Thiazolyl blue (Carl Roth) solution in 1xPBS. The plates were incubated for 3 h at 37°C. Then the solution was removed, and 25 μl DMSO was added into each well. Absorbance at 570 nm was measured in the Tecan Infinite 200 Pro reader.
4
Quantifying Cell Viability and Proliferation
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24 hours post transfection, 30 × 103 cells per well were re-seeded into 12-well plates. After additional 48 h of incubation, cells were incubated for 1.5 h with MTT-containing medium prepared by dissolving thiazolyl blue (Carl Roth, Karlsruhe, Germany). The cells were then solubilised using 10% Triton X-100 (Carl Roth)/0.1 M HCl (Fisher Scientific, Schwerte, Germany) in isopropanol (Sigma-Aldrich, Taufkirchen, Germany) and the extinction was then measured at 570 nm on a plate reader. For the cell survival assay, 10 × 103 cells were seeded per well in seven 24-well plates and transfected with siRNAs next day. Over a period of 7 days, MTT assays were performed and cell survival was plotted as relative cell number by measuring the MTT extinction coefficient for each day.
5
Cell Proliferation Assays: BrdU and MTT
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For BrdU incorporation assays using the Cell Proliferation ELISA Kit (Roche Diagnostics, Mannheim, Germany), 5 000 to 7 500 cells were reseeded 24 h post-transfection into a ViewPlate-96 Black cell culture plate (Perkin Elmer, Waltham, MA, USA). Following over-night culturing, cells were incubated 4–6 h with BrdU containing medium. After fixation for 1 h, cells were stained for 1.5 h with the peroxidase-conjugated anti-BrdU antibody, chemiluminescence substrate added and the emitted light quantified with a Centro LB 960 luminometer (Berthold Technologies GmbH, Stuttgart, Germany).
For MTT assays, 20 000 to 25 000 cells were reseeded 24 h after transfection in 24-well cell culture plates. After additional culturing for 24 h or 48 h, supernatant was replaced by MTT-containing medium (thiazolyl blue, Carl Roth GmbH) and plates were incubated for 2 h at 37°C. Medium was replaced by solubilization solution (10% Triton-100, 0.1 M hydrochloric acid in 2-propanol). Extinction was measured at 570 nm with the Multiskan FC photometer (Thermo Scientific, Schwerte, Germany).
For MTT assays, 20 000 to 25 000 cells were reseeded 24 h after transfection in 24-well cell culture plates. After additional culturing for 24 h or 48 h, supernatant was replaced by MTT-containing medium (thiazolyl blue, Carl Roth GmbH) and plates were incubated for 2 h at 37°C. Medium was replaced by solubilization solution (10% Triton-100, 0.1 M hydrochloric acid in 2-propanol). Extinction was measured at 570 nm with the Multiskan FC photometer (Thermo Scientific, Schwerte, Germany).
6
Cell Proliferation Assay Protocol
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To determine proliferation rates, cells were seeded on 6-well plates at a density of 1 × 105 cells per well. Cell viability was determined at 1, 3, and 6 days after seeding using the MTT assay by adding 90 µl of 5 mg/ml thiazolyl blue ≥ 98% (Carl Roth; 4022) to each well. The medium was aspirated after 1 h, and stained cells were dissolved in 400 µl of lysis buffer (80% isopropanol, 10% 1 M HCl, 10% Triton X-100). Absorption was measured at 595 nm using a plate reader. All values were normalized to day 1 to compensate for variations in seeding density. The mean value of three biological replicates was determined.
7
Cytotoxicity Assay of ZPT and Compounds
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N2a cells (3000 cells per well) were distributed into a transparent 384-well plate. The medium of each well was replaced by 50 μl of fresh medium the next day containing the indicated concentrations of ZPT, ZnCl2, or pyrithione. After incubation for 3 hours, the medium in each well was replaced by 50 μl of Thiazolyl blue (0.5 mg/ml; Carl Roth) solution in 1× PBS. The plates were incubated for 3 hours at 37°C. Then, the solution was removed, and 30 μl of DMSO was added into each well. Absorbance at 570 nm was measured in a Tecan Infinite M200 PRO plate reader.
8
Quantifying TNBC Cell Viability
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100μl of the indicated TNBC cell lines re-suspended at 30'000 cells/ml (100'000/ml for HCC-38 or 250'000/ml for IOWA-1T) were added into each well of a transparent 96-well plate. The cells were maintained in DMEM (DMEM/F12 in case of IOWA-1T) containing 10% FBS and incubated at 37°C, 5% CO 2 overnight. The medium of each well was replaced by 100μl fresh medium the next day containing the indicated concentrations of clofazimine. In case of combination experiments, the cells were first covered with 50µl of medium, and desired concentrations of clofazimine and doxorubicin in the well were created by adding them in 25µl of the corresponding pre-dilutions in the DMEM. After incubation for 3-4 days, the medium in each well was replaced by 100μl of 0.5 mg/ml Thiazolyl blue (Carl Roth) solution in 1xPBS. In case of IOWA-1T, which is a suspension cell line, 75μl of 1.5mg/ml Thiazolyl blue solution was added directly in the 150μl of the medium. The plates were incubated for 3h at 37°C. Then the solution was removed and 50μl DMSO was added into each well. In case of IOWA-1T, the plates were centrifuged at 1000g for 1min and the supernatant was carefully removed prior to addition of DMSO. Absorbance at 570nm was measured in a plate reader (Victor3, PerkinElmer).
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