实验材料
Dulbecco’s PBS (DPBS)
0.05% (w/v) trypsin in DPBS
DMEM
DMEM supplemented with 10% fetal bovine serum (DMEM/FBS)
Tumor necrosis factor-α (Sigma T0157), 10μg/ml solution in PBS
containing 1mg/ml BSA
Bright-Glo Luciferase Assay System (Cat.# E2610)
HEK 293 cells
实验流程
Day 1: Plate Cells
1. Grow HEK 293 cells in DMEM/FBS to approximately 75% confluency.
2. Harvest cells via trypsinization: Remove the DMEM/FBS, wash the cells with DPBS
and add the trypsin/DPBS (1X volume). After 2 minutes, add a 4X volume of
DMEM/FBS, collect the cell suspension and pellet the cells by centrifugation.
Aspirate the supernatant and resuspend in DMEM/FBS. We have routinely used a
concentration of 15,000 viable cells/100μl DMEM/FBS.
3. Dispense 100μl of the cell suspension into the wells of a 96-well plate. Plate enough
wells to perform each test condition in triplicate.
4. Cover the plate and place it in a tissue culture incubator at 37°C overnight (or for
24 hours).
Day 2: Transfect Cells
1. Transfect the cells using a high-efficiency transfection reagent. Each well of 96-well
plate to be transfected requires 0.1μg pGL4.32[luc2P/NF-κB-RE/Hygro] plasmid
DNA. Transfection conditions may require optimization.
2. Cover the plate and place it in a tissue culture incubator at 37°C overnight or as
needed for cell recovery depending on the transfection method used. We have used
24 hours recovery time for lipid-mediated transfections.
Day 3: Induce Transfected Cells
1. Prepare 1X induction and 1X control solutions. Calculate the volume of 1X induction
and 1X control solution by multiplying the number of wells needed for each solution
by 100μl and prepare 110% of this amount.
1X induction solution: Dilute 10μg/ml TNFα solution to 20ng/ml in DMEM/FBS.
Final TNFα concentration will be 20ng/ml.
1X control solution: DMEM/FBS.
2. Remove media from wells that will be treated with either 1X induction solution or
1X control solution.
3. Add 100μl of 1X induction solution to the cells to be induced and 100μl of
1X control solution to the control noninduced cells.
4. Return the plate to the tissue culture incubator and induce for 5 hours.
5. Analyze luciferase activity using an appropriate luciferase detection assay. We have
observed comparable results for fold induction of the vector using a variety of
luciferase reagents, including: Bright-Glo Luciferase Assay System (Cat.# E2610,
Technical Manual #TM052); ONE-Glo Luciferase Assay System (Cat.# E6110,
Technical Manual #TM292); Dual-Luciferase Reporter Assay System (Cat.# E1910,
Technical Manual #TM040); and Dual-Glo Luciferase Assay System (Cat.# E2920,
Technical Manual #TM058).
6. Calculate the fold induction as follows:
Fold Induction = Average relative light units of induced cells/Average relative light units of control cells
