Amplite® Luciferase Reporter Gene Assay Kit *Maximized Luminescence*

Protocol summary

1. Prepare cells (samples) with test compounds (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
2. Add equal volume of Luciferase Sensor working solution
3. Incubate at room temperature for 10 - 20 minutes
4. Monitor luminescence intensity at 560 nm

Important notes
Thaw all the kit components to room temperature before use. For all luminescent experiments, it is recommended to use white plates to get the best results.

1. For Cat.# 12518, transfer the whole content of Reaction Buffer (Component B) into the bottle of Luciferase Sensor (Component A) and mix well to make Luciferase Sensor working solution. 

2. For Cat.# 12519, add 10 mL of Reaction Buffer (Component B) and for Cat.# 12520, add 100 mL of Reaction Buffer (Component B) into the bottle of Luciferase Sensor (Component A) and mix well.  Then, transfer the resulted solution back to the bottle of Reaction Buffer (Component B).  Multiple washes are necessary to completely transfer the contents. Note: The reconstituted Luciferase Sensor working solution is not stable. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Run Luciferase assay:

1. Treat cells (or samples) with test compounds by adding 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in desired compound buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
   
   
2. Incubate the cell plate in a 5% CO₂ incubator at 37°C for desired period of time, typically 4 hours to overnight.
   
   
3. Add 100 µL (96-well plate) or 25 µL (384-well plate) per well of Luciferase Sensor working solution.
   
   
4. Incubate the plate at room temperature for 10 - 20 minutes. Keep from light.
   
   
5. Monitor luminescence intensity with a luminometer.

Establish standard Luciferase calibration curve: Note: Luciferase standard curve should be generated together with the above assay if the absolute amount of Luciferase in samples needs to be calculated.

1. Make a series dilutions of Luciferase in PBS buffer with 0.1% BSA by including a sample without Luciferase (as a control) for measuring background luminescence. Note: Typically Luciferase concentrations from 1 pg/mL to 1 ng/mL are appropriate.
   
   
2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of diluted Luciferase solution into an empty plate.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Luciferase working solution.
   
   
4. Incubate the reaction mixture at room temperature for 10 - 20 minutes, protected from light.
   
   
5. Record the luminescence intensity with a standard luminometer.
   
   
6. Generate the Luciferase standard curve.