Individual slices were placed in a 48-well plate with four hundred microliters of DMEM medium and eighty microliters of MTS assay reagent (Promega, Fitchburg, WI, USA). MTS is a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. MTS is bioreduced by cells into a formazan product that is soluble in tissue culture medium. The absorbance of the formazan product at 490 nm can be measured directly from 96-well assay plates without additional processing. The conversion of MTS into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes found in metabolically active cells. Reactions were incubated at 37 °C for 1 h with shaking. The OD490 nm of the supernatants was measured, using a Synergy H1 microplate reader (Biotek, Shoreline, WA, USA).
Mts assay reagent
Manufactured by Promega
Sourced in United States
The MTS assay reagent is a colorimetric method used to measure the metabolic activity of cells. It is a versatile tool for cell proliferation, cytotoxicity, and viability studies.
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Lab products found in correlation
Synergy h1 microplate reader, by Agilent Technologies (1 mentions)
Prism 6, by GraphPad (1 mentions)
Caco 2 cell line, by American Type Culture Collection (1 mentions)
Vancomycin hydrochloride, by GoldBio (1 mentions)
Linezolid, by Selleck Chemicals (1 mentions)
Fusidic acid, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Trypticase soy broth tsb, by BD (1 mentions)
Dimethyl sulphoxide dmso, by Merck Group (1 mentions)
Ethanol, by Thermo Fisher Scientific (1 mentions)
Trypticase soy agar, by BD (1 mentions)
Mannitol salt agar, by BD (1 mentions)
Mueller hinton broth (mhb), by Merck Group (1 mentions)
Clindamycin, by Merck Group (1 mentions)
Mupirocin, by AppliChem (1 mentions)
Auranofin, by Enzo Life Sciences (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Mts assay protocol, by Promega (1 mentions)
5 protocols using mts assay reagent
1
MTS Assay for Cell Viability
2
Cytotoxicity Evaluation of Compounds 1 and 2
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Compounds 1 and 2 were assayed (at concentrations of 16 μg/mL, 32 μg/mL, 64 µg/mL, and 128 μg/mL) against a human epithelial colorectal (Caco-2) cell line (American Type Culture Collection, ATCC HTB-37) using the MTS assay [26 (link)]. Cells were cultured in DMEM supplemented with penicillin-streptomycin, nonessential amino acids (1%), and FBS (10%), at 37 °C with CO2 (5%). The cells were incubated with compounds (in triplicate) or DMSO (negative control) in a 96-well tissue culture-treated plate at 37 °C with CO2 (5%) for two hours. The MTS assay reagent (Promega, Madison, WI, USA) was subsequently added to each well and plates were incubated for four hours at 37 °C with CO2 (5%). The quantity of viable cells (at OD490) after treatment was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data was analyzed via a two-way ANOVA, with post hoc Dunnet’s multiple comparisons test (n = 3, P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).
3
Antimicrobial Agents and Bacterial Strains
4
Cell Proliferation Assay Using MTS
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Cells were suspended in RPMI-1640 medium containing 10% FCS and 2% streptomycin/penicillin. After counting cells, they were resuspended (0.125 × 105 cells/mL) and 100 μL of cells were added to the center of the wells of a 96-well flat bottom culture plate. The medium was carefully aspirated on days 1, 3, 5, or 7 and replaced with 100 μL RPMI-1640 medium containing 10% FCS and 2% streptomycin/penicillin containing 20 μL of MTS assay reagent (Promega). Proliferation was determined by the formation of a colored formazan product. Plates were read at 490 nm using a plate reader. The Olympus fluorescent microscope was used at each required time point to image cells. The MTS assay result was supported by cell count data in some experiments.
5
Cytotoxicity Assay of ADOTA-M, DAOTA-M2, and TOTA
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Cells were seeded at a density of 5 × 103 per well of a 96-well plate (50 μl). After 24 h, compounds were added at the appropriate concentration in triplicate (total volume 100 μl). After a further 24 h, 20 μl of the MTS Assay reagent was added according to the Promega MTS Assay protocol40 . Absorbance at 490 nm (cisplatin, ADOTA-M and DAOTA-M2 ) or 510 nm (TOTA ) was recorded 2–4 h after the addition of the reagent. In the case of ADOTA-M and DAOTA -M2 , cell treatments were corrected for compound absorbance by subtracting the compound-only control run in parallel. Absolute IC50 was determined from the equation IC50=IC100+IC0/2, where IC0 is the absorbance of only media and IC100 is the absorbance of DMSO-only-treated cells. Three independent repeats were performed.
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