The inhibition of cell proliferation by cisplatin was evaluated through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. Cells were seeded in 96 well microplates at a density of 10,000 cells so that the well could have exponentially growing cells. After 24 h, cells were treated with cis-Pt at concentrations ranging from 0.1 to 100 µM and incubated for 72 h. For the test, cells were incubated with 0.5 mg/mL MTT for 1 h at 37 °C. Blue formazan formed after MTT precipitation was dissolved in DMSO, and optical density at 595 nm was evaluated in a microplate reader interfaced with Microplate Manager/PV version 4.0 software (BioRad, Hercules, CA, USA). GraphPad Prism software version 6.0 (Graphpad, Boston, MA, USA) was used to calculate the half-maximal inhibitory concentration (IC50).
Microplate manager pv version 4
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Microplate Manager/PV version 4.0 is a software application developed by Bio-Rad for managing and analyzing data from microplate-based assays. The software provides core functions for data acquisition, analysis, and reporting.
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4 protocols using microplate manager pv version 4
1
Cisplatin Inhibition of Cell Proliferation
2
Auranofin Cytotoxicity Evaluation in A2780 Cells
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Auranofin (Sigma Aldrich Merck) was dissolved in DMSO (Dimethyl sulfoxide) to obtain a stock solution of 20 mM.
Standard MTT (4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) proliferation assay was used to determine the effect of AF on A2780 cell viability. Exponentially growing cells were seeded in 96-well microplates in RPMI 1640 supplemented with 10% FCS at a density of 8x103. After 24 h, 0.7 μM of Auranofin (corresponding to the previously calculated 72-h-exposure IC50-dose) [11 (link),40 (link)] was added in a fresh RPMI medium and incubated for 6, 12, 24, 48 and 72 h. At the end of incubation, cells were treated for 1 h at 37 °C with 0.5 mg/ml MTT dissolved in PBS. Then, MTT was removed, cells were washed in PBS and 100 μl of stop solution (DMSO) were added. After 15 min of incubation at 37 °C, optical density was read in the microplate reader interfaced with the software Microplate Manager/PV version 4.0 (Bio-Rad Laboratories) at 595 nm. Cell death in the experiments with C501S TRAP1 mutant was determined after 24 h of AF treatment by flow cytometry (FACS Celesta, BD) after Propidium Iodide (Sigma-Aldrich) staining, measuring the SubG1 cell fraction.
Standard MTT (4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) proliferation assay was used to determine the effect of AF on A2780 cell viability. Exponentially growing cells were seeded in 96-well microplates in RPMI 1640 supplemented with 10% FCS at a density of 8x103. After 24 h, 0.7 μM of Auranofin (corresponding to the previously calculated 72-h-exposure IC50-dose) [11 (link),40 (link)] was added in a fresh RPMI medium and incubated for 6, 12, 24, 48 and 72 h. At the end of incubation, cells were treated for 1 h at 37 °C with 0.5 mg/ml MTT dissolved in PBS. Then, MTT was removed, cells were washed in PBS and 100 μl of stop solution (DMSO) were added. After 15 min of incubation at 37 °C, optical density was read in the microplate reader interfaced with the software Microplate Manager/PV version 4.0 (Bio-Rad Laboratories) at 595 nm. Cell death in the experiments with C501S TRAP1 mutant was determined after 24 h of AF treatment by flow cytometry (FACS Celesta, BD) after Propidium Iodide (Sigma-Aldrich) staining, measuring the SubG1 cell fraction.
3
Cytotoxic Effects of Gold Compounds on Cancer Cells
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The inhibition of cell proliferation by Au2phen, Auoxo6, and AuL12 on A2780 cell line was evaluated through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. Viable cells can convert MTT into formazan, which is coloured and detectable at 595 nm. Exponentially growing cells were seeded in 96 well-microplates at a density of 8 × 103 for 24 h and then gold compounds were added in fresh RPMI medium at concentrations ranging from 0.003 to 100 µM and incubated for 72 h. On the day of the test, cells were treated with 0.5 mg/mL MTT for 1 h at 37 °C. Following precipitation, blue formazan was dissolved in DMSO, and the optical density was read in a microplate reader interfaced with Microplate Manager/PV version 4.0 software (BioRad Laboratories, Hercules, USA). From the absorbance measurements, the half-maximal inhibitory concentration (IC50) value of each compound on A2780 cells was calculated using GraphPad Prism software version 6.0 (Graphpad Holdings, LLC, USA). The same protocol was applied on non-tumor cell lines, i.e., HEK and PNT1 cells.
The effects of these 72 h exposure IC50 doses on A2780 cell viability were also evaluated using an MTT time course assay at 12, 24, 48, and 72 h of drug exposure. The experimental protocol described above was applied.
The effects of these 72 h exposure IC50 doses on A2780 cell viability were also evaluated using an MTT time course assay at 12, 24, 48, and 72 h of drug exposure. The experimental protocol described above was applied.
4
MTT Cytotoxicity Assay for A2780 Cells
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Cell viability was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, 1 × 104 exponentially growing A2780 cells were seeded in 96 well-microplates for 24 h, then treated with tested compounds 1, 4 and 5 concentrations ranging from 0.003 to 100 µM and incubated for 72 h at 37 °C in a humidified incubator. On the day of the test, cells were treated with 0.5 mg/ml MTT for 1 h at 37ºC. Following precipitation, blue formazan was dissolved in DMSO, and optical density was read at 595 nm in a microplate reader interfaced with Microplate Manager/PV version 4.0 software (BioRad Laboratories). From Absorbance measurements, half-maximal inhibitory concentration (IC50) values of each compound were calculated by using GraphPad Prism Software 6.0.
The effects of these calculated 72-h exposure IC50 doses on A2780 cell viability were also evaluated using an MTT time course assay at 24, 48, and 72 h of drug exposure. The experimental protocol described above was applied. All MTT experiments were performed in triplicate (biological replicates) and in each assay all gold compound concentrations were tested in triplicate (technical replicates).
The effects of these calculated 72-h exposure IC50 doses on A2780 cell viability were also evaluated using an MTT time course assay at 24, 48, and 72 h of drug exposure. The experimental protocol described above was applied. All MTT experiments were performed in triplicate (biological replicates) and in each assay all gold compound concentrations were tested in triplicate (technical replicates).
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