Haynes:Luciferase

<- Back to Protocols

Cell Lysate Luciferase Assay

Principle: 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

1. Grow cells in a 6-well culture plate in standard growth medium.
2. When cells are 90 - 100% confluent, there will be enough cells for the following processing steps.
3. Harvest cells by standard trypsinization.
4. Pellet the cells at 1000 rpm for 3 min. Resuspend in 2 mL FACS buffer (10% FBS in 1x PBS).

LUCIFERASE ACTIVITY ASSAY

1. Prepare enough complete luciferase assay buffer for 3 replicates for all samples, 100 uL per replicate.
2. Filter 600 μL cells from each 2 mL experimental sample through strainer caps.
3. 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.
4. Load plain PBS into three wells as a background control.
5. Very quickly mix (by pipetting) 100 uL luciferase activity buffer into each 100 uL cell sample.
6. Incubate the reaction at room temperature for the recommended time.
7. Use the 96-well plate reader to measure the chemiluminescent signal. Three reads is recommended to capture output at maximum or steady state.

The current recommended program for reading luciferase activity on the Synergy H1 reader is:

• Protocol: KAH_luciferase
• Optimized specs: Luminescence, Endpoint, Full plate, Integration time 0:01.00 (MM:SS.ss), Filter set 1, Emission: full light, Optics: top, Gain 200, Read Speed: normal, Delay: 100 msec, Extended Dynamic Range, Read Height: 1 mm

CELL COUNTING

1. Use the Wang lab's Accuri flow cytometer. Be sure to check if anyone is using the cytometer while you plan to use it.
2. Set machine to read 20 uL of cells.
3. Set up the software to create a histogram of forward scatter vs. cell count.
4. Place the tube of strained cells onto the sample holder and run the sample.
5. Gate the data to include only only the highest peak of cells.
6. Record the cell count per 20 uL in a note book.
7. "Clean" the cytometer with a non-cell water sample. When the cytometer reads close to zero events in water, it is ready to run the next cell sample.
8. Repeat the previous three steps for the remaining
9. Do the following calculation: luciferase per cell = Luciferase signal - background signal / 5 x cell count for 20 uL. This is your final data.