Renilla-Firefly Luciferase Dual Assay Kit 

MCE Renilla-Firefly Luciferase Dual Assay Kit contains high-purity D-Luciferin, coelenterazine and proprietary reaction buffer. The Firefly Luciferase Working Solution is first added to the sample. This reagent contains D-Luciferin which can produce Firefly luciferase bioluminescence. Next, the Renilla Luciferase Working Solution is added to the same sample. It quenches the Firefly luciferase bioluminescence and produce Renilla luciferase bioluminescence. The light production of both reactions can be conveniently measured on a luminometer.

-20℃, 1 year

Protecting from light

1. Material Preparation

1.1 Preparation of Lysis Buffer (1×)

Dilute Lysis Buffer (5×) 1:5 with dH₂O (e.g. 1 mL Lysis Buffer + 4 mL dH₂O)

1.2 Preparation of Firefly Luciferase Working Solution

Briefly centrifuge tube of Firefly Luciferase Substrate. Prepare the Firefly Luciferase Working Solution by resuspending the lyophilized Firefly Luciferase Substrate in 10 mL Firefly Luciferase Buffer.

1.3 Preparation of Renilla Luciferase Working Solution

Briefly centrifuge tube of Renilla Luciferase Substrate (125×). Prepare the Renilla Luciferase Working Solution by adding 80 μL Renilla Luciferase Substrate (125×) to 10 mL Renilla Luciferase Buffer. Mix well by inverting the tube several times.

Note: Firefly Luciferase Working Solution and Renilla Luciferase Working Solution can be dispensed into aliquots. When protecting from light and avoiding repetitive freeze-thaw cycles, the reagent is stable for one month at -20°C or for one year when stored at -80°C.

2. Cell Lysis

2.1 Determine transfection parameters (plated cell density and subsequent incubation time) to ensure that cells are no more than 95% confluent at the desired time of lysate preparation.

2.2 Carefully remove culture medium from cells. Wash cells with wash solution, such as PBS, normal saline orserum-free culture medium, to remove residual medium.

2.3 Add proper volume of Lysis Buffer (1×). The recommended volumes of Lysis Buffer (1×) to add per well are as follows:

Note: The volume of added Lysis Buffer (1×) can be adjusted proportionally.

2.4 Add proper volume of Lysis Buffer (1×). The recommended volumes of Lysis Buffer (1×) to add per well are as follows:

2.5 Place the culture plates on a shaker with gentle shaking at room temperature for 15 minutes.

2.6 After lysis, centrifuge at 12,000 rpm for 10 minutes at 4 °C. Transfer the supernatants to a new tube for further analysis.

3. Luciferase Dual Assay

3.1 Add 20μL of cell lysate to a black flat-bottomed 96-well plate.

3.2 Add 100 μL of Firefly Luciferase Working Solution into each well containing cell lysate.

3.3 Immediately after adding the reagent, read the sample to capture the Firefly luciferase signals.

3.4 Add 100 μL of Renilla Luciferase Working Solution into each well from step 3.3, then immediately read the sample again to capture the Renilla luciferase signals.

3.5 Calculate the ratio of luminescence from the Firefly luciferase to the Renilla luciferase.

Note: 1) Assure Firefly Luciferase Working Solution and Renilla Luciferase Working Solution are equilibrated to room temperature before use.

2) The luminescence values of Firefly and Renilla Luciferase should be subtracted from the corresponding background values.