Leeporter™ Luciferase Assay Reagent- 1000 Test

1) Substrate (100X) Reconstitution:

 

Add 0.5 ml Substrate Reconstitution Solution to lyophilized Substrate vial and dissolve thoroughly by gently pipetting up and down, and/or inverting tubes. The reconstituted 100X substrate may be stored in a dark environment at -20^oC for up to one month.

 

2) Preparation of complete Assay Solution (for 96-well plate format):

 

Prepare complete Assay Solution fresh for each use, which should be used within 2 hours. Thaw 10 ml Assay Buffer and equilibrate to room temperature. Calculate how much complete assay solution is needed (Note: 50 ul of complete assay solution is required for each well of 96-well plate). Aliquote proper amount of Assay Buffer in a 15 ml tube and add corresponding amount of reconstituted 100X substrate to make a final 1X substrate assay solution. 

 

3) Luciferase Assay (96-well plate format):

1. Plate you target Leeporter™ Luciferase reporter cells in a white solid-bottom 96-well microplate (Note: The 96-well plate used should be compatible with the luminometer being used.) at 100 ul cells/well, based on the corresponding protocol to your target Leeporter™ cell line (Note: Each target Leeporter™ cell line protocol can be found in its corresponding Data Sheets.).

2. Stimulate or treat your target cells based on the corresponding protocol to your target Leeporter™ cell line.

3. After completion of stimulation/treament of your target cells, equilibrate the 96-well plate containing your target cells being assayed to room temperature for 10 minutes (Note: Do NOT remove or disturb cell culture medium.).

4. Using a multi-channel pipettor, add 50 ul complete Assay Solution directly to each plate well (Note: Do NOT remove cell culture medium when adding the Assay Reagent. So the Assay Reagent of 50 ul should be directly added on top of the cell culture of each well.).

5. Use automix for 2-3 seconds, and then read the plate in a luminometer within 1-5 minutes. (An example of a luminometer set up: SpectraMaxL (Molecular Devices): target wavelength of 470nm, integration time at 0.5 sec and automix for 2 sec).