LeeporterTM Luciferase Assay Reagent- 1000 Test

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 -20oC 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 -20oC 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 μl 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 you target LeeporterTM 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 LeeporterTM cell line (Note: Each target LeeporterTM cell line protocol can be found in its corresponding Data Sheets.) . [Total volume per well is 100 uL] 2. Stimulate or treat your target cells based on the corresponding protocol to your target LeeporterTM cell line. [Total volume per well becomes 105~110 uL = 100 ul cells + 5~10 ul treatment] 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.) . [Total volume per well becomes 155~160 uL = 100 ul cells + 5~10 ul treatment + 50 μl 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) .