p53 reporter luciferase assays

A p53-responsive reporter plasmid containing 13 copies of the p53-binding consensus sequence, pG13-LUC, was a gift from B. Vogelstein (Addgene plasmid no. 16442). Cells were transfected with pG13-LUC plasmid, and luciferase assays were carried out.
 

Materials and Reagents

1. NCI-H1299 Lung cancer cells (ATCC, catalog number: CRL-5803 )

2. PG13-luc (wt p53 binding sites) (Addgene plasmid no. 16442).

3. Lipofectamine™ 3000 Transfection Reagent （ Invitrogen™ catalog number: L3000008）

4. Opti-MEM™ I Reduced Serum Medium （Gibco™ catalog number: 31985062）

5. 24-Well Cell-Culture Treated Multidishes (Thermo Scientific™ Nunc™ catalog number: 142475)

6. Pipette tips (USA Scientific, catalog numbers: 1110-3000, 1110-1000, 1111-2021)

7. T75 flask (Corning, Falcon®, catalog number: 353136)

8. 100 mm TC-treated Tissue Culture Dish (Corning, Falcon®, catalog number: 353003)

9. 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352099)

10. 96-well plates (Greiner Bio One International, catalog number: 650185)

11. 96-well white OptiPlate (Perkin Elmer 6002291)

12. Dulbecco’s phosphate-buffered saline (DPBS) (Corning, catalog number: 21-031- CM)
13. Trypsin EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056)

14. Dual-Luciferase Assay System (Catalog # E1960, Promega)

15. Fetal bovine serum (Corning, catalog number: 35-010-CV)

16. DMEM–high glucose (Sigma-Aldrich, catalog number: D6429-500ML)

17. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081)

18. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122)
 

Equipment

1. Pipettes (Eppendorf, model: Z740362-6EA,2231000862)

2. Tissue culture hood. (NUAIRE,NU-534)

3. VWR® Air Jacketed CO₂ Incubators

4. Microcentrifuge (Eppendorf, model: 5424)

5. Table-top centrifuge (Eppendorf, model: 5810)
 

Procedure

A. Grow low passage H1299 cells from liquid nitrogen stock

1. Briefly thaw cells at 37 °C water bath.

2. Pipette cells into 15 ml thermal scientific Nunc conical tubes and spin for 5 min at 1,500 x g. Aspirate media, re-suspend cells in 15 ml growth media and plate in T75 culture flask (cells are plated at the density at 1 x 10⁶ and are cultured at 37 °C with 5% CO₂).

3. Passage cells every 3–4 days to ensure that they do not enter senescence. Cell transfection should only be carried out between passages 5 and 25 after thawing. Plate cells for transfection only 1 day before the experiment. The day before transfection, dissociate cells that are 80–90% confluent in a T 75 flask. Count the cells using standard trypan blue exclusion. The cell culture must have >90% viability and be 80% confluent on the day of transfection.
 

B. Transfection

1. Day 0: Seed cells (8.0x 104 cells) in a total volume of 500 μl complete growth media (DMEM/10% FBS).

2. Day 1: On the day of transfection, perform the following steps, which have been optimized for a single well of a 24-well plate using Invitrogen™ Lipofectamine™ 3000 Transfection Reagent:

Culture the cells in DMEM supplemented with 10% (v/v) fetal bovine serum at 37 °C under a humidified 5% CO₂ atmosphere.

C. Luciferase reporter assay

1. After 48 hours post-transfection, remove the medium from the cultured cells by vacuum pump attached by tubing to a liquid trap and a gel loading pipette tip, and gently apply a sufficient volume (500 μl/well) of PBS to rinse the bottom of each well.

2. Dispense 100 μl of 1x PLB (see Recipes) into each well and gently shake the culture plate for 15 min at room temperature.

3. Transfer 20 μl PLB lysate without cell debris to each well of a 96 well OptiPlate.

4. Program the GloMax® 96 Microplate Luminometer to perform a 2-sec premeasurement delay, followed by a 10-sec measurement period for each reporter assay.

5. Dispense 50 μl LAR2 substrate (see Recipes) into each well of the 96-well OptiPlate, mix by pipetting 2 or 3 times, and measure Firefly luciferase activity using GloMax® 96 Microplate Luminometer. The Luminescence reads represent the Firefly luciferase activity.

6. Dispense 50 μl Stop & Glo® Reagent (see Recipes) into the same plate, mix by pipetting 2 or 3 times and measure the Renilla luciferase activity by GloMax® 96 Microplate Luminometer. The Luminescence reads represent the Renilla luciferase activity.

7. Relative activity =Firefly luciferase activity/Renilla luciferase activity
 

Recipes

1. 1x passive lysis buffer (PLB)

Add 1 volume of 5x PLB from Dual-luciferase reporter assay kit to 4 volumes of distilled water and mix well.
The 1x PLB can be stored at 4 °C no more than one month

2. LAR2 substrate (from Dual-luciferase reporter assay kit)

Resuspend the lyophilized Luciferase Assay Substrate in Luciferase Assay Buffer 2 (10 ml for one bottle) and store at -20 °C less than 1 month or -70 °C less than 1 year

3. Stop & Glo® Reagent (from Dual-luciferase reporter assay kit)

Freshly add 1 volume Stop & Glo® Substrate to 49 volumes Stop & Glo® Buffer and vortex 10 sec.