To observe osteoclast differentiation, RAW264.7 cells were cultured in 96‐well plates in medium containing various concentrations of rosiglitazone in the presence 50 μg·mL−1 of RANKL. After 5 days, cells were stained for TRAP with a commercial kit (TRACP & ALP double‐stain kit, Takara, Shiga, Japan). Cytotoxicity of rosiglitazone was assayed using an MTT reagent (Amresco, Inc., Solon, OH, USA). In this case, cells were treated for 1 or 5 days without RANKL addition.
Mtt reagent
Manufactured by Avantor
Sourced in United States
The MTT reagent is a colorimetric assay used to measure the activity of enzymes that reduce the MTT tetrazolium compound to its insoluble formazan, providing a quantitative assessment of cell viability and proliferation.
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Lab products found in correlation
Multilabel plate reader, by PerkinElmer (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Avantor (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Tracp alp double stain kit, by Takara Bio (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Cy3 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master rox, by Roche (1 mentions)
Elisa kit, by Cusabio (1 mentions)
Cck 8 solution, by Dojindo Laboratories (1 mentions)
Cell light edu apollo 567 in vitro imaging kit, by RiboBio (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Norcantharidin nctd, by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate dcfda, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Flat bottom 96 well plate, by Greiner (1 mentions)
Synergytm ht plate reader, by Agilent Technologies (1 mentions)
Pitavastatin, by Selleck Chemicals (1 mentions)
30 protocols using mtt reagent
1
Rosiglitazone Modulates Osteoclast Differentiation
2
Assessing Cell Proliferation and Colony Formation
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After transfection with POH1 siRNAs or Flag (3 × Flag)-tagged POH1 plasmid and scramble siRNA or vector plasmid, the cells were seeded in each well of 96-well plates (3 × 104 cells/ml) in 100 μl medium and cultured for 5 days. The proliferation assay was performed by treating cells with 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (5 mg/ml, AMRESCO, Solon, OH) for 4 h at 37°C. The formazan crystals formed were dissolved in dimethyl sulfoxide (150 μl/well). The absorbance of each sample was measured using a multilabel plate reader (PerkinElmer) at 490 nm wavelength. For the colony formation assay, 500 cells were seeded into six-well plates and incubated at 37°C in a humidified atmosphere containing 5% CO2 for 10-14 days. Colonies were fixed with methanol, stained with 0.1% crystal violet, and counted.
3
MTT Assay for Cytotoxicity Evaluation
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Cells were seeded at a density of 1.5 × 104 cells in each well of 24-well plates and treated with erlotinib or KYA1797K for 0, 24, 48, and 72 hours. MTT reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AMRESCO) was diluted in RPMI 1640 at a concentration of 0.5 mg/mL and was treated with cells for 2 hours at 37 °C. Media was removed and insoluble purple formazan was solubilized with 1 mL DMSO for 30 minutes. The absorbance of formazan product was determined at 590 nm. Each experiment was repeated three times. In order to acquire IC50 values of KYA1797K treatment in different cell lines, the same procedure was performed except for the difference in cell density and treatment time. NCI-H1650, PC-9, NCI-H460, NCI-H23, and A549 cells were seeded at a density of 8.0 × 103, 1.0 × 104, 8.0 × 103, 6.0 × 103, and 1.5 × 104 cells in each well of 96-well plates, respectively, and the all cells were treated with KYA1797K for 72 hours.
4
Cell Proliferation Assay with MTT
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MDA-MB-231 cells, MCF-7, SKBR3 and BT474 cells were seeded into 96-well plate at 2,500 cells and 5,000 cells per well, respectively. Transient transfection was performed on day-1 (24 hours prior to MTT assay). To measure cell proliferation, the cells were incubated with MTT reagent (AMRESCO, Solon, OH, USA) for 2 to 4 hours on day 0, day 1, day 2, day 4 and day 6. The absorbance at 570nm was recorded on the indicated days. Each assay was performed in triplicate.
5
Evaluating miR-1277 Effects on HepG2 Cell Proliferation
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After transfection, HepG2 cells at a density of 3,000 cells/well were plated in 96-well plates. At 24, 48, 72, and 96 h after transfection of miR-1277 mimic, miR-1277 inhibitor, scramble RNA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-liumbromide (MTT) reagent (AMRESCO, Solon, OH, USA) was added into each well, and the cells continued to be incubated for 4 h at 37°C. The supernatants were then removed, and dimethyl sulfoxide (DMSO; 150 μl/well; Sigma-Aldrich, St. Louis, MO, USA) was added to dissolve the formazan crystals. The absorbance of each sample at 490 nm was then measured using a multilabel plate reader (PerkinElmer, Waltham, MA, USA). In addition, at 72 h after transfection, the colony formation assay was also performed to assess cell proliferation. Cells at a density of 100 cells/dish were placed into 60-mm culture dishes and maintained in complete medium for 2 weeks. Colonies were then fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich). Colonies that contained more than 30 cells were then counted under a microscope (IX83; Olympus, Tokyo, Japan). All determinations were conducted in triplicate.
6
Synergistic Effects of Drug and Heat
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The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to confirm the synergistic effect of concurrent drug and heat treatment. AGS cells (1 × 10⁴ cells/mL) were plated on a 6-well plate, treated with 0, 10, 15, and 20 μg/mL of AR for an hour, and then exposed to 37 or 43 °C for 30 min. Twenty microliters per well of MTT reagent (2 mg/mL in phosphate-buffered saline; PBS) (AMRESCO, Solon, OH, USA) were added after 48 h of incubation. The wells were then incubated at 37 °C for 2 h in the dark. Following suction, 100 μL of DMSO was added to each well, the mixture was agitated, and the absorbance at 570 nm was determined using a spectrophotometric plate reader. Relative cell viability was standardized against untreated controls.
7
MTT Assay for HepG2 Cytotoxicity
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HepG2 cells were plated at a density of 5 × 103 cells/well in 96-well plates in 100 μL of medium. After si-HMGB3 or miR-200b, mimics were transfected for 24, 48, 72, and 96 hours, 5 mg/mL MTT reagent (T0793; AMRESCO, Solon, OH) was added to each well and the plates were incubated at 37°C for a further 4 hours. Then, the MTT was extracted with 150 μL dimethyl sulfoxide (DMSO [Sigma-Aldrich, St. Louis, MO]) and the absorbance was recorded at 490 nm with a spectrophotometer. Each assay was performed using 5 replicates per sample in 3 independent experiments.
8
MTT Proliferation Assay for Cell Viability
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Cell growth and viability was measured using the MTT proliferation assay. Briefly, 2000 cells were seeded in 96-well plates in 200 μl RPMI 1640 containing 10% FBS with or without 2 nM R1881 treatment. Cell growth was examined 7 days later. Before testing, 20 μl of MTT reagent (2.5 mg/ml MTT in PBS, Amresco Inc. Solon, Ohio) was added and the cells were incubated for a further 4 h at 37°C. Then 250 μl of dissolving reagent DMSO (Amresco Inc.) was added to dissolve the formazan crystals. The optical density (OD) was measured at wavelength of 490 nm on a microplate reader.
9
Paraquat and Tacrolimus Modulate TGF-β1 Signaling in Fibrosis
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Paraquat and tacrolimus were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. High glucose DMEM and FBS were obtained from HyClone; Cytiva. MTT reagent was purchased from Amresco, LLC. Rat TGF-β1 (cat. no. CSB-E04727r), SMAD3 (cat. no. CSB-EL021788RA), SMAD7 (cat. no. CSB-E09225r) and connective tissue growth factor (CTGF; cat. no. CSB-E07876r) levels were analyzed using ELISA kits purchased from Cusabio Technology LLC. BSA (cat. no. A8020), the TGF-β1 receptor type I/II dual inhibitor (LY2109761; cat. no. ab29286) and anti-SMAD3 antibody (cat. no. ab40854) were obtained from Abcam, and the anti-SMAD7 antibody (cat. no. 42-0400) was purchased from Thermo Fisher Scientific, Inc. The Cy3 conjugated Goat Anti-Rabbit IgG secondary antibody (cat. no. GB21303) was obtained from Wuhan ServiceBio Technology Co., Ltd. RNA extracting fluid (cat. no. G3013) was obtained from Wuhan ServiceBio Technology Co., Ltd. HyPure™ Molecular Biology Grade Water (cat. no. SH30538.02) was obtained from HyClone. RevertAid First Strand cDNA Synthesis kit was obtained from Thermo Fisher Scientific, Inc. FastStart Universal SYBR Green Master (Rox) was obtained from Roche Diagnostics. Primers were obtained from Wuhan ServiceBio Technology Co., Ltd.
10
Cell Viability and Colony Formation Assay
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Transfected cells were seeded in 96-well plates (2–4 × 104 cells/mL) with 100 µL medium in each culture for 5 days. The diffusion test was performed by adding 20 µL of MTT reagent (5 mg/mL, AMRESCO, Solon, OH, USA) for 4 hours at 37°C. Then, the formazan crystals were dissolved in dimethyl sulfoxide (150 µL/well). The absorbance values (OD 590 nm) were measured by a multilabel plate reader (PerkinElmer). For the colony formation assay, 500 cells were seeded onto six-well plates with 2 mL DMEM per well. After 10 days of culture, cell colonies were fixed with methanol and then stained with 0.1% crystal violet, and the number of colonies was counted by microscope.
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