Reporter Gene Assay : Dual Luciferase assay
Cell Culture and Transfection
Day 1 (Monday), AM
1. Distribute 2 ml of cells into each well of 6-well culture plate 2. Incubate the cells for 24 h in CO incubator 2
Day 2 (Tuesday), PM
3. Prepare plasmid DNA (10 ;g of target LUC plasmid [Firefly luciferase] + 0.5 ;g of
pRL-SV40 control vector [Renilla luciferase] / 0.5 ml Opti-MEM I) and transfection
reagent (Lipofectin, 1 mg/ml, InVitrogen, 84 ;g [84 ;L] / 0.5 ml Opti-MEM I) in
separate tubes, and allow to stand at room temperature for 30 – 45 min
4. Combine two solutions, mix gently, and incubate at room temperature for 10 – 15 min
； “Transfection solution”
5. Wash cells once with serum-free growth media
6. Add 1 ml of transfection solution prepared above to each well 7. Incubation for 1.5 h at 37 ？C ； “Transfection”
8. Remove transfection solution from each well, add 2 ml of complete growth media (w/
serum) into each well and incubate the cells for 24 h in CO incubator ； “Recovery 2
Day 3 (Wednesday), PM
9. The cells are washed with HANKS and experimental growth media with chemical
treatments are replenished
10. Incubation for additional 16 h in CO incubator 2
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Day 4 (Thursday), AM
Preparation of cell extracts and enzyme assay
1. Add 4 volumes of distilled water to 1 volume of 5X Passive Lysis Buffer (Dual-
？Luciferase Reporter Assay System, Promega #E1960) to produce a 1X Passive Lysis
Buffer (“1X PLB”).
[ 1.3 mL of 5X PLB + 5.2 mL of D.W. for 12 wells ]
2. Remove the medium from the cells to be assayed. Wash the cells with 1X PBS. 3. Add a sufficient volume of 1X PLB to cover the cells (500 ;L for each well of a 6-
well plate). Rock the plate slowly several times to ensure complete coverage of the
4. Rock the culture plate at room temperature for 15 min.
5. Transfer the cell lysate to a microcentrifuge tube and centrifuge at 12,000 x g for 1
min at 4 ？C.
6. Transfer the supernatant (cell extract) to a fresh tube.
7. The extracts may be assayed directly or stored at -70？C.
8. Prepare Luciferase Assay Reagent II (“LAR II”) by resuspending the provided
lyophilized Luciferase Assay Substrate in 10 mL of the supplied Luciferae Assay
Buffer II. [ 100 ;L reagent per assay ]
？9. Preparation of Stop & Glo Reagent :
？1) Transfer 200 ;L of Stop & Glo Substrate Solvent into the amber glass vial
？ containing dried Stop & Glo Substrate and mix well (“50X” stock solution).
？2) Add 1 volume of reconstituted 50X Stop & Glo Substrate to 50 volumes of Stop
？？ & Glo Buffer in a glass tube (“Stop & Glo Reagent”)
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？？ [ 50 ;L of 50X Stop & Glo Substrate + 2.5 mL of Stop & Glo Buffer in glass
tube for 12 samples]
10. Measure the luminescence using a Luminometer with dual injector system.
Volume of PLB lysate : 10 ;L
Injector #1 : LARII, 100 ;L ； Firefly luciferase (target plasmid) Injector #2 : Stop & Glo Reagent, 100 ;L ； Renilla luciferase (control plasmid) Sensitivity : 45 – 65%
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