To begin, generate each reporter substrate and check their concentration using a spectrophotometer. Obtain 200 nanograms of reporter substrate. Electrophorese each reporter substrate alongside a high molecular weight DNA ladder in a 0.7%agarose TAE gel containing a fluorescent double-stranded DNA stain.
Verify that defined bands of the correct size are observed for reach reporter substrate. If assessing the impact of compounds on the reporter assay, dispense compounds dissolved in the vehicle into the wells of a 96-well plate. To prepare the transfection mix for a 96-well plate, dilute the Firefly control plasmid and NanoLuc double strand break repair reporter substrate in 500 microliters of transfection buffer in a 1.5-milliliter tube.
After adding the lipid base transfection reagent, vortex the DNA mixture briefly, and incubate for 10 minutes at room temperature. To harvest the cells, trypsinize and re-suspend them in fresh medium containing 10%fetal bovine serum, or FBS. Then, determine the cell count, and re-suspend three times 10 to the power of six cells in 8.5 milliliters of medium with FBS in a fresh 15-milliliter tube.
Add the DNA transfection mix into the cell suspension and invert the tube several times to mix. Plate 80 microliters of cell suspension per well, and incubate the 96-well plate at 37 degrees Celsius with 5%carbon dioxide for 24 hours. Prepare the control luciferase and the reporter luciferase reagent according to the manufacturer's instructions.
Add substrate into an appropriate volume of assay buffer at a one-to-100 ratio and mix. After the plate and reagents reach room temperature, add 80 microliters of control Firefly luciferase reagent per well. Shake the plate for three minutes on an orbital shaker at 450 revolutions per minute.
Place the plate in the luminescence plate reader, and measure the control luciferase luminescence signal. To measure the reporter luciferase signal, add 80 microliters of NanoLuc reporter luciferase reagent per well. After a three-minute incubation in an orbital shaker at 450 revolutions per minute, leave the plate to rest at room temperature for seven minutes.
Finally, measure the reporter luciferase luminescence signal in the luminescence plate reader. Evaluation of the component NanoLuc and Firefly luminescence signals showed that the observed repaired defect was driven by a reduction in the NanoLuc's signal, while the Firefly control signal was unperturbed.
