D-Luciferin (sodium)

D-luciferin sodium 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]. We of er the firefly luciferase (HY-P1004), luciferin free acid (HY-12591A), as well as its water-soluble sodium salts (HY-12591) and potassium salts (HY-12591B) . In Vitro:1. Precautions
a) The D-luciferin salts are readily soluble in aqueous buffers (pH 6.1-6.5) up to 100 mM. Stock solutions can be made in ATP-free water and stored at -20°C, protect from light. The free acid must be neutralized with an appropriate base to solubilize. At a higher pH, luciferin undergoes a base-catalyzed formation of dehydroluciferin, as well as racemization to the L-isomer.
b) The D-luciferin can be used with any existing reporter assay or ATP assay system.
c) If testing for ATP, minimize all possible sources of ATP contamination by wearing gloves and using ATP-free containers. Use only sterile ATP-free water and reagents. Use autoclaved water for all reagent preparations.
2. Experimental Protocols
This protocol only provides a guideline, and should be modified according to your specific needs.
The following protocol is an example for potassium and sodium salt preparation. It can be adapted for most cell types and in vivo animal use.
2.1 Example protocol for in vitro bioluminescent image assays
a) Prepare a 100 mM (100-200X) Luciferin stock solution in sterile water. Mix well. Use immediately, or make single use aliquots, and store at -20°C, avoid freeze-thaw cycles, avoid exposure to the light.
b) Prepare a 0.5-1 mM working solution of D-Luciferin in pre-warmed tissue culture medium.
c) Aspirate media from cultured cells.
d) Add Luciferin working solution to cells, and incubate the cells for 5-10 minutes at 37°C just prior to imaging.
2.2 Example protocol for in vivo bioluminescent image assays
a) Prepare a 15 mg/mL Luciferin stock solution in DPBS, without Mg²⁺ and Ca²⁺. Mix well.
b) Filter sterilizes the solution through a 0.2 μm filter. Use immediately, or make single use aliquots, and store at -20°C, avoid freeze-thaw cycles, avoid exposure to the light.
c) Inject the luciferin intra-peritoneally (i.p.) 10-15 minutes before imaging at 150 mg/kg (or 10 μL/g of luciferin stock solution) of the animal body weight.
Note: A kinetic study of luciferin should be performed for each animal model to determine peak signal time.
2.3 Example protocol for luciferin reporter assays
a) Prepare a 100 mM Luciferin stock solution in sterile water. Use immediately, or make single use aliquots, and store at -20°C, avoid freeze-thaw cycles, avoid exposure to the light.
b) Prepare a 1 mM working solution of D-Luciferin with 3 mM ATP, 1 mM DTT and 15 mM MgSO4 in 25 mM tricine buffer pH 7.8.
c) Pipette 5-10 μLof cell lysate into a microplate. Use lysis reagent or buffer without lysate as a blank.
d) Prime luminometer with luciferin working solution according to manufacturer’s instructions.
e) Inject 200 μL of luciferin working solution with no delay and a 10 second integration time. In Vivo:Bioluminescence imaging (BLI) using firefly luciferase (Fluc) as a reporter gene and D-luciferin as a substrate is currently the most widely used technique. The total signal intensity is plotted against the time after D-luciferin injection to generate a time-intensity curve. In addition to the peak signal, the signal at fixed time points (5, 10, 15, and 20 minutes) after D-luciferin injection is determined as a surrogate for the peak signal. The signal in a given time-intensity curve is normalized to the peak signal in the curve to represent the temporal pattern of change after D-luciferin injection^[3].
Inject 10 μL of D-luciferin (intraperitoneally or intravenously) per gram of body weight stock solution: a standard 150 mg/kg injection is typically about 200 μL for a 20 g mouse.
Thaw D-luciferin (potassium or sodium salt) at room temperature and dissolve in dPBS (without calcium or magnesium) to a final concentration of 15 mg/mL. Wet the 0.22 μM filter with 5-10 mL of sterile H₂O and discard the water. Sterilize the D-luciferin solution through the prepared 0.22 μM syringe filter.
Injection into the body for 10-20 min (wait for the light signal to reach the strongest stable plateau period) before imaging analysis.
Note: It is recommended to establish a luciferase kinetic curve for each animal model to determine the time of maximum signal detection and signal plateau period. After luciferin injection into the mouse, it can diffuse throughout the mouse body in about 1 min.
For intraperitoneal injection, the diffusion is slower, the onset of luminescence is slower, and the duration of luminescence is longer.
For tail vein injection of luciferin, the diffusion is faster, the onset of luminescence is faster, but the duration of luminescence is shorter.