Experimental
procedures
Cells from each cell lines were plated into 96-wells as 10,000 cell/well. The day after, five concentrations of the novel small molecules (synthetic 1,2,4-triazole[3,4-b]-1,3,4-thiadiazine derivatives) dissolved in DMSO and only-DMSO, as negative control, were added in duplicate. The plates were incubated at 37oC in a humidified incubator with 5% CO2 for different time intervals.
Since the cell lines used are adherent, at the end of each incubation period wells were washed with 1xPBS gently and then fixed with 10% TCA (trichloroacetic acid) for 1 hour at 4oC. Termination of fixation was followed by staining each well by SRB (Sulforhodamine B) for 10 minutes. Washing with 1% acetic acid several times removed the excess dye and the protein-bound SRB was dissolved in 10mM Tris base solution for OD determination at 515 nm via microplate reader.
For the Kinase Assay cells were plated with 60% confluency to 6-wells and on the next day were treated with the calculated IC50 and IC100 concentrations of our putative kinase inhibitors. After exact 24 hour incubation at 37oC in a humidified incubator with 5% CO2, cells were scraped and lysed respectively.
According to the protein concentration of cell lysates determined by Bradford Assay, the Kinase Reaction Buffer and cell lysates were initiated a reaction to give a final volume of 40µL.
ATP-Detection Reagent supplied from Lonza was added and incubated at room temperature for 10 minutes before reading the luminescence of each sample.
Cells from each cell lines were plated into 96-wells as 10,000 cell/well. The day after, five concentrations of the novel small molecules (synthetic 1,2,4-triazole[3,4-b]-1,3,4-thiadiazine derivatives) dissolved in DMSO and only-DMSO, as negative control, were added in duplicate. The plates were incubated at 37oC in a humidified incubator with 5% CO2 for different time intervals.
Since the cell lines used are adherent, at the end of each incubation period wells were washed with 1xPBS gently and then fixed with 10% TCA (trichloroacetic acid) for 1 hour at 4oC. Termination of fixation was followed by staining each well by SRB (Sulforhodamine B) for 10 minutes. Washing with 1% acetic acid several times removed the excess dye and the protein-bound SRB was dissolved in 10mM Tris base solution for OD determination at 515 nm via microplate reader.
For the Kinase Assay cells were plated with 60% confluency to 6-wells and on the next day were treated with the calculated IC50 and IC100 concentrations of our putative kinase inhibitors. After exact 24 hour incubation at 37oC in a humidified incubator with 5% CO2, cells were scraped and lysed respectively.
According to the protein concentration of cell lysates determined by Bradford Assay, the Kinase Reaction Buffer and cell lysates were initiated a reaction to give a final volume of 40µL.
ATP-Detection Reagent supplied from Lonza was added and incubated at room temperature for 10 minutes before reading the luminescence of each sample.