Cytotoxicity assays were carried out as described previously (19 (link)). In short, MDBK cells, exponentially grown in the 96-well plates (approximately 5 × 103 cells/well), were incubated in 5% CO2 at 37°C for 24 h. Then, the medium was removed and serial dilutions of compounds were added. DMSO was used as a negative control. The cells were proliferated at 37°C for 3 days, and their overall metabolic activity was determined by the method of MTS/PMS (Promega, USA). The 50% cytotoxic concentration (CC50) was calculated using a non-linear regression fitting of the data as the compound concentration necessary to reduce 50% cell viability compared to control non-treated cells.
Mts pms
Manufactured by Promega
Sourced in United States
The MTS/PMS is a compact, flexible and versatile laboratory equipment used for performing various analytical and experimental tasks. It combines the functionality of a microplate reader and a plate spectrophotometer, allowing for the measurement of absorbance, fluorescence, and luminescence in microplate formats. The core function of this product is to provide researchers with a reliable and accurate tool for a wide range of applications, including cell-based assays, enzyme activity measurements, and other biochemical analyses.
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Lab products found in correlation
M5 microplate reader, by Molecular Devices (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Elisa reader, by Bio-Rad (2 mentions)
Facsaria sorter, by BD (1 mentions)
Safire2, by Tecan (1 mentions)
Renilla luciferase assay system, by Promega (1 mentions)
Elx800 microplate reader, by Agilent Technologies (1 mentions)
Rhtnf α, by R&D Systems (1 mentions)
Transwell plate, by Corning (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Anisomycin, by Selleck Chemicals (1 mentions)
Spectrostar nano reader, by BMG Labtech (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Agno3, by Merck Group (1 mentions)
Haucl4, by Merck Group (1 mentions)
Nabh4, by Merck Group (1 mentions)
Celltiter 96 kit, by Promega (1 mentions)
Simplicity sims 00001 equipment, by Merck Group (1 mentions)
27 protocols using mts pms
1
Cytotoxicity Assay for Cell Viability
2
Cytotoxicity Evaluation of CDV
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Cytotoxicity of CDV was evaluated by the MTS method. Briefly, cells were seeded in 96-well plates at 10,000 cells per cm2. After overnight incubation at 37°C, CDV (10, 20, 50 or 100 μg/ml) was added for 24 h. Next, the cells were washed and incubated with fresh medium containing 10% FBS for another 24, 48 or 120 h. To determine the 50% cytotoxic concentration (CC50), the medium was removed and 90 μl medium plus 10 μl MTS/PMS (Promega) were added to each well. After an incubation period of 2 h at 37 °C, the optical density of each well was determined at 498 nm in a microplate reader.
3
Gal8-Expressing RAW 264.7 Macrophage Generation
4
ZIKV Infection Effect on hPSC-HLCs and Huh7 Cells
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hPSC-HLCs and Huh7 cells were seeded and differentiated in a 96-well format. On day 16 of hepatocyte differentiation, cells were infected with ZIKV. Six days post infection, medium was removed from the ZIKV-infected hPSC-HLCs and Huh7 cells. 100μL 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazinemethosulfate (MTS/PMS; Promega, Leiden, The Netherlands) was added to the cells. After 1h30min incubation at 37°C in a 5% CO2 humidified incubator, the optical density (OD) was determined at 498nm using the Tecan Safire2 TM. The % cell survival was calculated using the following formula: % survival = 100 x (ODinfected/ODuninfected).
5
Cellular Toxicity Evaluation of Flavones
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Cellular toxicity for the tested compounds was evaluated using a previously reported cell viability assay [43] (link). The cells were harvested during the log phase of growth and inoculated onto 96-well plates at a final concentration of 3×103 cells per well. After 24 h of incubation at 37°C under 5% CO2, the cell cultures were treated with flavones at concentrations of 1, 10, 100, and 200 µM. Twenty microliters of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate (MTS/PMS; Promega, Madison, WI) were added to each well, and the absorbance at 490 nm was measured according to the manufacturer's recommendations. The mean value was obtained from four replicate wells. A control group treated with 0.1% DMSO was evaluated simultaneously.
6
Virus Infectivity and Cytotoxicity Assay
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For single cycle virus infections, virus was added to subconfluent cell layers and allowed to adsorb for 1 hour, after which virus was removed and fresh (drug-containing) medium was added to the cells. At 8 h p.i., cells were subjected to three cycles of freeze-thawing after which virus titers were determined by endpoint titration. Alternatively, cells were lysed to determine the intracellular Renilla luciferase activity with the Renilla Luciferase Assay System (Promega). For multicycle virus infections, cells grown to confluency in 96-well plates were subjected to serial dilutions of the compound and inoculated with the appropriate virus. After 3 days of incubation, cell viability was measured: after removal of the medium, 10% MTS/PMS (Promega) was added to each well and quantified spectrophotometrically at 498 nm in a microplate reader. The 50% effective concentration (EC50) was defined as the concentration of compound that inhibited virus-induced cytopathic effect formation by 50% and was calculated using logarithmic interpolation.
7
Antiviral Activity of Compounds Against BVDV
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The activity of compounds against BVDV was achieved by inhibiting the cpBVDV C24V strain induced cytopathogenicity in MDBK cells. Cells were seeded in 96-well plates and incubated overnight in growth medium in 5% CO2 at 37°C. Then, the cells were infected with virus at 103 TCID50/mL, using a multiplicity of infection (MOI) of 0.05. After 2 h, the virus was removed and maintenance medium (DMEM supplemented with 2% FBS) with or without serial dilutions of compounds was added. Concentrations of compounds were from 6.25 to 200 μg/mL. DMSO was used as a control sample. The cells were incubated at 37°C for 3 days. Subsequently, the antiviral activity was evaluated through the viral titration and the cytopathic effect (CPE) inhibition, by MTS/PMS (Promega, USA). The 50% effective concentration (EC50) was calculated using a non-linear regression fitting of the data as the compound concentration necessary to reduce 50% cytopathic effect on MDBK cells compared to DMSO treated control cells.
8
Cytostatic Effect Evaluation of Inhibitor
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For the assessment of the potential cytostatic effect of the evaluated inhibitor, cells were seeded at a density of 6.5 × 103 cells per well of a 96-well plate in complete DMEM. Serial dilutions of the test compounds in complete DMEM were added 24 hr after seeding. Cells were allowed to proliferate for 3 days at 37°C, after which the cell number was determined by means of the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)/phenazinemethosulfate (MTS/PMS) method (Promega). The CC50 (value derived from the dose-response curve) represents the concentration at which the metabolic activity of the cells would be reduced to 50% of the metabolic activity of untreated cells. The selectivity index, indicating the therapeutic window of the compound, was calculated as the CC50/EC50.
9
Neutralization Assay for CMAB008 Evaluation
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Assays for the neutralization of TNFα cytotoxicity were used to compare the biological activity of CMAB008 and reference product. TNFα-sensitive L929 cells (1.5×104/well, ATCC CCL1) were seeded in a 96-well culture plate and incubated in 100 µL RPMI 1640/DMEM supplemented with 10% new bovine serum and cultured overnight (18–24 hours) at 37°C, 5% CO2. Serial dilutions of the Abs to be tested for neutralization were prepared in medium containing 20 µg/mL dactinomycin and 4.9 ng/mL rhTNFα (R&D Systems). Consequently, 100 µL prepared serially diluted test samples were added to each well of a 96-well tissue-culture plate in duplicate. After 14–16 hours of incubation at 37°C, 5% CO2, 20 µL MTS/PMS (Promega) solution was added before another 1–4 hours of incubation. Absorbance was determined with a BioTek ELx800 microplate reader at 490 and 630 nm. The following equation established from a four-parameter logistic model was used to calculate EC50: Y=(A-B)/(1+[X/C]D)+B, where X represents the concentration of CMAB008 in the samples, Y absorbance at 450 nm, D the slope of the logit-log plot, C EC50, and A and B maximal and minimal absorbance, respectively. The calculated value of each sample was required to be within the range of the standard curve.
10
Analysis of Cellular Proliferation and Migration
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For MTS analysis, stable shRNA expression cells (shLacZ, shMTHFD2#50, shMTHFD2#53, shPAICS#74, shPAICS#75, and shMTHFD2/PAICS) were seeded in 96-well plate at 4000 cells per well with DMEM containing 2 μg/ml puromycin. At each 24 h, 48 h, 72 h time point, 20 μl of MTS/PMS (Promega) were added and incubated for 2 h at 37 °C with 5% CO2. The cell viability was determined by OD 490 nm using an ELISA reader (BioRad). For colony formation assay, the stable shRNA expression cells were seeded in 6-well plates (600 cells/well) with DMEM containing 2 μg/ml puromycin and incubated for 2 weeks. After 2 weeks, the colonies were fixed with methanol overnight and then stained by 4% GIEMSA stain for 1 h and analyzed by ImageJ. For cell migration, 3 × 104 stable shRNA expression cells with 1% FBS medium were loaded into the inserts, and medium containing 10% FBS was loaded into the lower compartments of an 8-μM pore size Transwell plate (Corning). The cells were incubated at 37 °C with 5% CO2 for 6 h, then fixed for 30 min with 100% methanol and stained with 1% GIEMSA for 30 min. Cotton swabs were used to remove cells from the upper side of the inserts. Images of five different microscope fields of each insert were captured and counted.
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