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D-luciferin potassium is the natural substrate of the enzyme luciferase (Luc) that catalyzes the production of the typical yellowgreen light of fireflies. The 560 nm chemiluminescence from this reaction peaks within seconds, with light output that is proportional to luciferase concentration when the substrate luciferin is present in excess. The luciferase (luc) gene is a popular reporter gene for research and agent screening. Chemiluminescent techniques are virtually background-free, making the luc reporter gene ideal for detecting low-level gene expression. As little as 0.02 pg of luciferase can be reliably measured in a standard scintillation counter. In addition to its role as a reporter of gene expression, luciferase is commonly used in an extremely sensitive assay for ATP. 

1. Preparation of cells: Prior to the experiment, inoculate the target cells expressing luciferase into a culture plate overnight or culture for several hours to allow them to adhere to the wall.

2. Prepare working solution: Dilute the storage solution with preheated complete culture medium to 150 µg/mL.

3. Add working solution: Remove the original culture medium of the cells and immediately add newly prepared complete culture medium containing 150 µg/mL D-Luciferin to each well before imaging analysis.

4. Incubation and testing:

Incubating cells at 37 ° C for a short period of time before testing can usually enhance signal intensity.

Suggest determining the optimal incubation time first: after adding the working solution, detect the signal every 10 minutes for 40 minutes, draw the kinetic curve, and find the peak signal time of the cell line. In general, a 10 minute incubation may be sufficient.

Preparation of working solution: DPBS is used to prepare a working solution with a concentration of 15mg/mL, and filtered through a 0.2 µm filter membrane for sterilization.

Calculate injection dose: The general administration dose is 150mg fluorescein/kg mouse body weight. Converted to a volume of 10 µL/g of mouse body weight.

Example: For a 20g mouse, 20g x 10 µL/g  =200 µL of working solution should be injected, containing 15 mg/mL x 0.2 mL=3 mg of D-Luciferin.

Injection: The most commonly used method is intraperitoneal injection (IP). Before injection, ensure that the solution is protected from light.

Imaging: 10-15 minutes after injection, anesthetize the mice and place them in a live imaging system (such as IVIS) for image analysis. Similarly, it is recommended to establish dynamic curves in advance for each animal model to determine its peak signal time.

Room temperature in continental US; may vary elsewhere.

2-8°C, sealed storage, away from moisture and light

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)