KeratinoSens Assay for Identifying Skin Sensitizers Step-By-Step
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The cells to be used for the 96-well assays are stored in a liquid nitrogen freezer. When ready for use, the cells are thawed, resuspended, and transfered to a culture flask, where they will grow and multiply until optimal conditions have been achieved.
Trypsin solution is added to the flask to remove the cells. The cells are resuspended in medium, counted, and “seeded” into 96-well plates. For each trial, 3 plates are seeded for the luciferase determination. One clear plate is seeded for the cytotoxicity determination.
Each test material is serially diluted to make a range of doses. Each plate contains a maximum of 7 test articles covering a range of 12 dilutions, a solvent control, a series of 5 dilutions of the positive control and 1 blank well.
Test Article Removal and Rinsing
The 3 plates designated for the luciferase endpoint are removed from the incubator and allowed to set at room temperature. The optimal temperature for luciferase enzymatic activity is room temperature.
The test materials are removed by decanting.
Only the plates designated for the luciferase endpoint are rinsed with a buffered saline solution to remove any residual test material. The plate designated for the MTT endpoint receives MTT without prior rinsing.
Addition of Reagents for Luciferase Endpoint
After the test materials are rinsed from the plates, a buffered saline solution is added to the 96-well plate, followed by the addition of a ONE-Glo reagent (Promega). The ONE-Glo reagent contains a lysing agent that breaks the cells open. In addition, the ONE-Glo contains luciferin and ATP.
The luciferase enzyme produced by the cells breaks down the luciferin, and in the presence of ATP, produces light.
Addition of Reagent for Cytotoxicity Endpoint
After the test material is removed from the plate, a MTT solution is added to all of the wells. The plate is incubated to allow the MTT (initially a yellow solution) to be reduced by microsomal enzymes into blue/purple insoluble form.
After the appropriate incubation time, SLS (a surfactant) is added to lyse the cells to release the MTT into solution. The plate is incubated overnight.
Luminescence: A luminometer (equipment used to measure luminescence), is used for the luciferase induction endpoint. The luciferase enzyme produced by the cells breaks down the luciferin, and in the presence of ATP, produces detectable light that is measured as Relative Light Units (RLUs).
The RLU values are then used to determine the amount of light detected from each well. These values are compared to the solvent controls to determine the EC 1.5 and Imax values.
Cytotoxicity: The 96-well plate that contains the reduced MTT is placed on a plate shaker to fully extract the MTT and evenly distribute the dye in each well. A spectrometer measures the absorbance of each sample at a specific wavelength.
The absorbance values (optical density) are then used to determine the viability of each well by comparing the optical density of each test material treated well to that of the solvent control wells to determine the IC50 values.