Haynes:Luciferase
Cell Lysate Luciferase Assay
Principal: Cells expressing the luciferase reporter gene are lysed to release the luciferase enzyme into solution. D-luciferin substrate is added to the solution. Free luciferase enzyme then catalyzes a reaction that generates light (bio/chemiluminescence). In this application, the light signal serves as a proxy for the amount of luciferase gene expression. Note that data on individual cells is lost using this procedure, but it is useful for measuring relative average levels of reporter gene activity in different cell samples.
Kits we use in the lab include:
- Biotium - Steady-Luc Firefly HTS Assay Kit #30028-1
Below is a general protocol to help you plan/ prepare for a luciferase assay. This protocol includes a cell counting step so that luciferase signal can be normalized as luciferase per cell. This way, differences in luciferase signal will not be an artifact of input variations.
CELL CULTURING & HARVESTING
- Grow cells in a 6-well culture plate in standard growth medium.
- When cells are 90 - 100% confluent, there will be enough cells for the following processing steps.
- Harvest cells by standard trypsinization.
- Pellet the cells at 1000 rpm for 3 min. Resuspend in 2 mL FACS buffer (10% FBS in 1x PBS).
LUCIFERASE ACTIVITY ASSAY
- Filter 600 μL cells through strainer caps.
- Transfer 3x 100 μL strained cells to 3 wells in 96-well plate (black walls; clear, flat bottom). Save remainder of strained cells on ice for flow cytometry/ counting.
CELL COUNTING
