The pGL4.37[luc2P/ARE/Hygro] Vector contains four copies of an antioxidant response element (ARE) that drives transcription of the luciferase reporter gene luc2P (Photinus pyralis). luc2P is a synthetically-derived luciferase sequence with humanized codon optimization that is designed for high expression and reduced anomalous transcription. The luc2P gene contains hPEST, a protein destabilization sequence, which allows luc2P protein levels to respond more quickly than those of luc2 to induction of transcription. The vector backbone contains an ampicillin resistance gene to allow selection in E. coli and a gene for hygromycin resistance to allow selection of stably transfected mammalian cell lines.
In this example protocol, the pGL4.37[luc2P/ARE/Hygro] Vector is used to measure activation of the ARE in HEK293 cells upon treatment with tert-Butylhydroquinone or D,LSulforaphane.The pGL4.75 Vector (encoding Renilla luciferase) is used as a normalization control. In designing such experiments, it is important that the chosen cell type can be transfected efficiently and that it expresses the proper components of the signaling pathway of interest in order to generate the biological response. Protocol optimization may be required for your particular cell type and assay conditions.
實驗材料
Dulbecco’s PBS (DPBS; Life Technologies Cat.# 14190)
0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
DMEM (Life Technologies Cat.# 11995)
complete medium (DMEM supplemented with 10% fetal bovine serum
[Life Technologies Cat.# 11995] and 1X NEAA [Life Technologies Cat.# 11140])
Charcoal-stripped FBS (Life Technologies Cat.# 126776-011)
DMSO (Sigma Cat.# D2650)
Opti-MEM I (Life Technologies Cat.# 31985)
FuGENE HD Transfection Reagent (Cat.# E2311)
tert-Butylhydroquinone (Sigma Cat.# 112941)
or D,L-Sulforaphane (Sigma Cat.# S4441)
Dual-Glo Luciferase Assay System (Cat.# E2940)
HEK293 cells
pGL4.75[hRluc/CMV] Vector (Cat.# E6931)
實驗流程
Day 1: Plate Cells
1. Grow HEK293 cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash
with DPBS and treat with one volume of 0.05% trypsin-EDTA. Resuspend cells in
four volumes of complete medium.
2. Quantify the cells and dilute to 1.5 × 105 cells/ml in complete medium.
3. Plate 100μl per well to a solid, white 96-well plate (Corning Cat.# 3917).
4. Incubate for 24 hours in a 37°C, 5% CO2 incubator.
Day 2: Transfection
1. Dilute pGL4.37[luc2P/ARE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase vector
constructs in a 10:1 mass ratio, respectively, to 12.5ng total DNA/μl in Opti-
MEM I.
2. Add FuGENE HD to a 3:1 lipid:DNA ratio. Mix by pipetting. Incubate at room temperature
for 20 minutes.
3. Add 8μl transfection complex per well (100ng DNA/well) and incubate for 18 hours
in a 37°C, 5% CO2 incubator.
Day 3: Medium Replacement and Cell Treatment
1. Remove medium from cells and replace with 72μl of DMEM + 0.5% charcoalstripped
FBS per well.
2. Incubate for 6 hours in a 37°C, 5% CO2 incubator.
3. Resuspend tert-Butylhydroquinone (tBHQ) to 500mM in ethanol. Serially dilute into
ethanol to give 1,000X stocks. Resuspend D,L-Sulforaphane to 200mM in DMSO.
Serially dilute into DMSO to give 1,000X stocks. Dilute the 1,000X stocks into Opti-
MEM I to give 10X stocks.
4. Add 8 μl of the 10X stocks of tBHQ or D,L-Sulforaphane to each well and incubate
for 18 hours in a 37°C, 5% CO2 incubator.
Day 4: Luciferase Measurement
1. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room temperature
for approximately 15 minutes.
2. Add 80μl of the Dual-Glo Luciferase Assay System detection reagents and measure
luminescence following the recommended protocol (Refer to the Dual-Glo
Luciferase Assay System Technical Manual, #TM058 for details).
