MRC-5 or Hep-2 cells were seeded at a density of 5 × 103/well in 96-well plates and treated the next day with 27OHC, 2HP-βCD:27OHC, or blank formulation at concentrations ranging from 0.07 to 150 μM to generate dose-response curves. Control samples (100% of viability) were prepared by treating cells with culture medium. After 72 h of incubation, cell viability was determined using a Cell Titer 96 Proliferation Assay Kit (Promega, Madison, WI, USA) and following the manufacturer's instructions. Absorbance was measured using a Microplate Reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA) at 490 nm. Viability of oxysterol-treated cells is expressed as a percentage relative to cells incubated with culture medium supplemented with equal volumes of ethanol.
Celltiter 96 proliferation assay kit
Manufactured by Promega
Sourced in United States
The CellTiter 96 Proliferation Assay Kit is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The kit measures the conversion of a tetrazolium compound, MTS, into a colored formazan product, which can be quantified using a spectrophotometer. This assay provides a simple, reliable, and quantitative way to assess cell proliferation or cytotoxicity in a 96-well format.
Automatically generated - may contain errors
Lab products found in correlation
Model 680 microplate reader, by Bio-Rad (6 mentions)
Prism software, by GraphPad (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Kaluza flow analysis software, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
7 aminoactinomycin d 7 aad, by Beckman Coulter (1 mentions)
Model 680, by Bio-Rad (1 mentions)
Multiskan fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Prism 5, by GraphPad (1 mentions)
18 protocols using celltiter 96 proliferation assay kit
1
Oxysterol Cytotoxicity Assay
2
Cell Viability Assay using MTS
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Cell viability was measured using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Cell cultures were seeded in 96-well plates were incubated with different concentrations of compounds in triplicate under the same experimental conditions described for the antiviral assays. Cell viability was determined using the CellTiter 96 Proliferation Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Absorbances were measured using a Microplate Reader (Model 680, BIORAD) at 490 nm. The effect on cell viability at different concentrations of the compound was expressed as a percentage, by comparing absorbances of treated cells with those of cells incubated with culture medium and equal volumes of vehicle. The 50% cytotoxic concentrations (CC50) and 95% confidence intervals (CIs) were determined using Prism software (Graph-Pad Software, San Diego, CA).
3
Cytotoxicity Evaluation of Polymer Samples
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BTHP1-DC-SIGN cells were incubated with the polymer samples for different time points (3 h and 30 min, 24 h, and 72 h) and then labelled with 7-aminoactinomycin D (7-AAD) (Beckman Coulter, Milan, Italy). Samples were acquired by using a Gallios™ Flow Cytometer and data were analysed with Kaluza® Flow Analysis Software (both from Beckman Coulter). HeLa and Vero cells were seeded at in 96-well plates; the next day, they were treated with serially diluted compounds. After 24 or 72 h of incubation, cell viability was determined using the CellTiter 96 Proliferation Assay Kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Absorbances were measured using a Microplate Reader (Model 680, BIORAD) at 490 nm.
4
Nanoparticle Cytotoxicity Evaluation
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The toxicity of
NPs was examined
using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Vero cell cultures (African green
monkey fibroblastoid kidney cells ATCC CCL-81) seeded in 96-well plates
were incubated in DMEM (Gibco-BRL, Gaithersburg, MD) supplemented
with 2% FBS with different concentrations of nanoparticles or cyclodextrins
for 24 h. Cell viability was determined using the CellTiter 96 Proliferation
Assay Kit (Promega, Madison, WI) according to the manufacturer’s
instructions. Absorbance was measured using a Microplate Reader (Model
680, BIORAD) at 490 nm. Cells incubated without nanoparticles nor
cyclodextrins were used as the negative control. The effect on cell
viability at different concentrations of nanoparticles or cyclodextrins
was expressed as a percentage of live cells, by comparing the absorbance
of treated cells with those of the cells incubated with the culture
medium. All experiments were performed in triplicates.
NPs was examined
using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Vero cell cultures (African green
monkey fibroblastoid kidney cells ATCC CCL-81) seeded in 96-well plates
were incubated in DMEM (Gibco-BRL, Gaithersburg, MD) supplemented
with 2% FBS with different concentrations of nanoparticles or cyclodextrins
for 24 h. Cell viability was determined using the CellTiter 96 Proliferation
Assay Kit (Promega, Madison, WI) according to the manufacturer’s
instructions. Absorbance was measured using a Microplate Reader (Model
680, BIORAD) at 490 nm. Cells incubated without nanoparticles nor
cyclodextrins were used as the negative control. The effect on cell
viability at different concentrations of nanoparticles or cyclodextrins
was expressed as a percentage of live cells, by comparing the absorbance
of treated cells with those of the cells incubated with the culture
medium. All experiments were performed in triplicates.
5
Cell Viability Assay of Milk Dilutions
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Cell viability was measured by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Confluent cell cultures seeded in 96-well plates were incubated with different dilutions of milk in triplicate under the same experimental conditions described for the antiviral assays. Cell viability was determined by the CellTiter 96 Proliferation Assay Kit (Promega) according to the manufacturer's instructions. Absorbances were measured using a Microplate Reader (Model 680, BIORAD) at 490 nm. Their effect on cell viability at different milk dilutions was expressed as a percentage, by comparing the absorbances of treated cells with that of the cells incubated with culture medium alone. The 50%-cytotoxic dilutions (CD50) and 95% confidence intervals (CIs) were determined with Prism 4 software.
6
Oxysterols Dose-dependent Cytotoxicity
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Cells were seeded at a density of 5 × 103/well in 96-well plates and treated the following next day with serially-diluted oxysterols (25HC, 27HC, 7αHC, 7βHC or 7κC) to generate dose-response curves. After 24 or 120 hours of incubation, cell viability was determined using the CellTiter 96 Proliferation Assay Kit (Promega, Madison, WI, USA), and following the manufacturer's instructions. Absorbances were measured using a Microplate Reader (Model 680, BIORAD) at 490 nm. The effect of oxysterols at different concentrations on cell viability was expressed as a percentage, by comparing absorbances of treated samples with those of the respective controls.
7
Cytotoxicity of Plant Extracts
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Cells were seeded at a density of 5 × 103/well in 96-well plates and treated the following day with serially-diluted methanol extracts of B. macrodonta, S. cryptantha or R. lanata (1.2 μg/ml to 1666 μg/ml) to generate dose–response curves. Control wells (100% of viability) were prepared by treating cells with equal volumes of methanol, corresponding to 6.7% (v/v) to 0.0048% (v/v) in cell media. After 24 h of incubation, cell viability was determined using the CellTiter 96 Proliferation Assay Kit (Promega, Madison, WI, USA), and following the manufacturer’s instructions. Absorbances of both treated (AbsT) and untreated (AbsMETH) samples were measured using a Microplate Reader (Model 680, BIORAD) at 490 nm. The % of cell viability was calculated according to the following formula: (AbsT X 100)/AbsMETH. The 50% cytotoxic concentration (CC50) was determined using logarithmic viability curves. Where possible, a selectivity index (SI) was calculated by dividing the CC50 by the EC50 value.
8
Cytotoxicity Evaluation of Drug Formulations
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To test the cytotoxic effect of the drug formulations, the MTS [3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay was carried out as described by Donalisio et al. [45 (link)]. Briefly, Vero cells were treated with serial concentrations of VACV or formulations ranging from 90 µM to 5.6 µM. Cell viability was assessed at 24 h post-treatment using the CellTiter 96 Proliferation Assay Kit (Promega, Madison, WI, USA) by comparing the absorbances of treated cells with those of untreated cells.
9
Assessing Colostrum's Cell Viability Impact
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Colostrum samples and extracellular vesicles were investigated for their impact on cell viability by means of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, as described in Cagno et al. (2015) [22 (link)]. HFF-1 cells, pre-seeded at a 5 × 103/well density in 96-well plates in DMEM 10% FBS, were challenged with serial dilutions of the aqueous fraction of colostra and extracellular vesicles. After 5 days of incubation at 37 °C, the cell monolayers were washed three times with DMEM, and cell viability was assessed with a Cell Titer 96 Proliferation Assay Kit (Promega, Madison, WI, USA). An aliquot of 20 microliters of MTS reagent was added to 100 µL DMEM per well; the cells were incubated at 37 °C for 4 h. Absorbance was measured at 491 nm using a Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA). The percentage of absorbances of the treated cells to the cells incubated with only a culture medium was calculated, and the 50% cytotoxic concentrations (CC50) and 95% confidence intervals (95% CI) were determined for the preterm colostra and colostrum-derived EVs using Prism software (Graph-Pad Software, San Diego, CA, USA).
10
Oxysterol Cytotoxicity in Cell Lines
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Vero and HeLa cells were seeded into 96-well plates at a density of 104 cells/well, and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. Oxysterols dissolved in ethanol (25 hydroxycholesterol 25HC, 27 hydroxycholesterol 27-HC, 7α hydroxycholesterol 7αHC, 7β hydroxycholesterol 7βHC and 7Ketocholesterol 7KC) were added to the cells at different concentrations ranging between 150 µM and 0.1 µM, with a replicate number of three wells per concentration. 25-HC was purchased from Sigma Aldrich (Saint Louis, Missouri, USA); 7αHC, 7βHC, 7KC and 27-HC were purchased from Avanti Polar Lipid (Alabaster, Alabama). After 24 or 48 h incubation period, cell viability was measured by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay by the Cell Titer 96 Proliferation Assay Kit (Promega, Madison, WI,USA) according to the manufacturer's instructions. Absorbance was measured using a Microplate Reader (Model 680, BIORAD) at 490 nm. The percent of viability was calculated in comparison with cells treated with equal volumes of ethanol. The 50% cytotoxic concentrations (CC50) and 95% confidence intervals (CI) were determined using GraphPad PRISM software (Graph-Pad Software, San Diego, CA).
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