For CCK analysis, lentiviral H2030 cells were treated with either 10-70 μM cisplatin (Sigma-Aldrich, USA) or 80-640 μg/ml carboplatin (Sigma-Aldrich, USA) for 24 h. siRNA and miRNA mimics transfected H2030 cells were treated with either 4-32 μM cisplatin or 30-240 μg/ml carboplatin for 48 h. Four thousand cells per well were plated on a 96-well plate in sextuplicate overnight. After drug treatment, cells were then treated with WST-8 Cell Counting reagent (Dojindo, Japan) for 1 h at 37 °C according to the manufacturer’s protocol. Analysis was performed using the Infinite M200 PRO Microplate Reader (Tecan) with 450 nm absorbance and the survival rate was calculated by normalizing untreated cells to 100%.
Wst 8 cell counting reagent
Manufactured by Dojindo Laboratories
Sourced in Japan
WST-8 cell counting reagent is a colorimetric assay for the quantification of viable cells. It utilizes a tetrazolium salt that is reduced by cellular dehydrogenases, producing a water-soluble formazan dye that can be measured spectrophotometrically. The amount of formazan dye generated is directly proportional to the number of living cells.
Automatically generated - may contain errors
Lab products found in correlation
Cisplatin, by Merck Group (1 mentions)
Carboplatin, by Merck Group (1 mentions)
Infinite m200 pro microplate reader, by Tecan (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Dojindo Laboratories (1 mentions)
Elisa plate reader model 680, by Bio-Rad (1 mentions)
3 protocols using wst 8 cell counting reagent
1
Cisplatin and Carboplatin Cytotoxicity Assay
2
In Vitro Cytotoxicity Assay for Pancreatic Cancer
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In vitro cytotoxicity assay: Pancreatic cancer cells (PANC-1, BxPC-3 or Capan-2), were seeded in 96-well plates at a density of 23,000 cells per well and incubated in a fresh DMEM, (Sigma-Aldrich) at 37 °C, 5% CO2 for 24 h. After rinsing with PBS, cells were subjected to the addition of NRM, NDM, or special media conditions. Serially diluted solutions of synthesized compounds (5.5% v/v DMSO in NDM) were added to the cells up to a series of concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM and 6.25 μM, followed by a 24 h incubation at 37 °C, 5% CO2. Cell morphology was monitored under an inverted microscope. Cytotoxicity was assessed on PBS washed cells by the addition of fresh DMEM containing 10% WST-8 cell counting reagent (Dojindo). Following a 3 h incubation at 37 °C, 5% CO2, absorbance values were measured with a plate reader at 450 nm, and cell viability was calculated using the equation:
At least two replicate experiments were conducted for each medium condition reported, and similar results were obtained.
At least two replicate experiments were conducted for each medium condition reported, and similar results were obtained.
3
Cell Proliferation Assay with siRNA
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Cells were seeded in 96-well plates at a density of 5,000 cells per well, followed by culturing at 37°C in a humidified 5% CO2 atmosphere after siRNA treatments: siRNA for IMP3 (siIMP3) and siRNA for negative control (siCtrl), as described above. After 24, 48, 72 and 96 h growth, cells were incubated with WST-8 cell counting reagent (Dojindo, Kumamoto, Japan) for 2 h at 37°C. The optical density of the culture solution in each well were determined at 450 nm using an ELISA plate reader (model 680, BioRad Laboratories, Hercules, CA, USA). Experiments were performed in triplicate.
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