Luciferase Assay Reagent

Overview

Kit Components

Application Note

Protocol:

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°C for up to one month.
2) Assay Buffer:
Thaw Assay Buffer of 50 ml in room temperature water bath and dissolve thoroughly any precipitates by swirling and/or gentle vortexing until solution becomes clear. Assay Buffer can be aliquoted (e.g. 5-10 ml each) and stored at -20°C for several months.
3) 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 Assay Buffer and equilibrate to room temperature with swirling and/or gentle vortexing. Calculate how much complete assay solution is needed (Note: 50 ul of complete assay solution is required for each well of 96-well plate). Aliquot 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.
4) Luciferase Assay (96-well plate format):
1. Plate your target 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 cell line (Note: Each target cell line protocol can be found in its corresponding datasheets.). [Total volume per well is 100 uL]
2. Stimulate or treat your target cells based on the corresponding protocol to your target cell line. [Total volume per well becomes 105~110 uL = 100 ul cells + 5~10 ul treatment]
3. After completion of stimulation/treatment 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.). [Total volume per well becomes 155~160 uL = 100 ul cells + 5~10 ul treatment + 50 ul complete assay solution]
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).

Validation Images & Assay Conditions

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