Natural substrate of luciferase (Luc) enzyme
D-luciferin (Firefly luciferin) targets firefly luciferase with a Km value of 5.7 μM (recombinant firefly luciferase) [1]
D-luciferin (Firefly luciferin) serves as the substrate for luciferase-mediated bioluminescent reaction (EC50=3.2 μM for maximal bioluminescence intensity) [2]

1. Note:
a) D-luciferin exists three forms: free acid, potassium salt, and sodium salt. The sodium and potassium forms of D-luciferin can be easily dissolved in aqueous buffer solutions (pH 6.1-6.5). The stock solution can be prepared with ATP free water and stored in the dark (protect from light) at -20 ° C. While D-luciferin free acid must be neutralized with an appropriate base in order to dissolve. At higher pH values, luciferin will form dehydro-luciferin under alkaline catalysis and racemize into L-luciferin.
b) D-fluorescein can be used in any existing report analysis or ATP analysis system.
c) If testing ATP, please wear gloves and use ATP free containers to minimize all possible sources of ATP contamination. Only use sterile ATP free water and reagents. Prepare all reagents using high-pressure sterilized water.
2. Experimental protocol: This protocol may be adjusted to meet your specific requirements, as it only serves as a guide.
2.1 Example of In vitro Bioluminescence Image Analysis.
a) Prepare D-luciferin stock solution in DMSO. Mix well. Immediately use or aliquot it, store at -20 ℃, avoid repeated freezing and thawing, and avoid exposure to light.
b) Prepare 0.5-1 mM D-Luciferin working solution and preheat the tissue culture medium.
c) Suck out the culture medium from the cultured cells.
d) Add D-luciferin working solution to the cells and incubate them at 37 ℃ for 5-10 minutes before imaging.
2.2 In vivo experiments
Due to the large dose used for in vivo experiments, generally 150 mg/kg, it is recommended to choose potassium salt or sodium salt of D-luciferin.
2.3 Example for Fluorescein Reporter Gene Testing
a) Prepare D-luciferin stock solution in DMSO. Immediately use or aliquot it, store at -20 ℃, avoid repeated freezing and thawing, and avoid exposure to light.
b) Prepare 1 mM D-Luciferin working solution and 3 mM ATP, dissolve 1 mM DTT and 15 mM MgSO4 in 25 mM tricine buffer at pH 7.8.
c) Transfer 5-10 μ L of cell lysate to a microplate. Use lysis reagents or buffer solutions without lysate as blank.
d) According to the manufacturer's instructions, infuse the luminescence meter with D-Luciferin working solution.
e) Immediately inject 200 μ L of D-Luciferin working solution, with an integration time of 10 seconds

---

In recombinant firefly luciferase-catalyzed reactions, D-luciferin (Firefly luciferin) (0.1–100 μM) exhibited concentration-dependent bioluminescence with a quantum yield of 0.88, and the reaction reached maximal intensity at 10 μM. The reaction rate constant (kcat) was 1.2×104 s-1 [1]
At pH 7.8 and 37°C, D-luciferin (Firefly luciferin) showed optimal bioluminescent activity; a pH decrease below 6.5 or temperature above 42°C reduced activity by >50%. Co-incubation with ATP (1 mM) enhanced luminescence intensity by 2.3-fold [2]
In luciferase-expressing HEK293 cells, D-luciferin (Firefly luciferin) (10–500 μM) induced dose-dependent bioluminescence, with an EC50 of 45 μM. The luminescence peaked at 30 minutes post-incubation and remained stable for 2 hours [2]
Compared to its analogues (e.g., 6'-aminoluciferin), D-luciferin (Firefly luciferin) showed 1.8-fold higher luminescence intensity in vitro, but a shorter half-life (120 min vs. 180 min for the analogue) [1]

The most popular method at the moment is bioluminescence imaging (BLI), which uses D-luciferin as a substrate and firefly luciferase (Fluc) as a reporter gene. A time-intensity curve was created by graphing the total signal intensity versus the amount of time following D-luciferin injection. Apart from the peak signal, a surrogate signal for the peak signal was identified at specific time intervals (5, 10, 15, and 20 min) following D-luciferin injection. To depict the pattern of temporal changes following D-luciferin injection, the signal in a given time-intensity curve is normalized against the peak signal in the curve [3]. Intraperitoneal or intravenous injection: Use 10 μL of D-Luciferin stock solution per gram of body weight. An injection of 150 mg/kg should typically contain 200 μL for a 20 g mouse. After thawing at room temperature, dissolve D-Luciferin (sodium or potassium salt) in dPBS (without calcium or magnesium) until the final concentration reaches 15 mg/mL. After passing 5–10 mL of sterile HO through a 0.22 µM filter, discard the water. Pass the D-luciferin solution through a 0.22 µM syringe filter that has been produced.

---

In nude mice bearing luciferase-expressing HCT116 xenografts, intraperitoneal injection of D-luciferin (Firefly luciferin) (150 mg/kg) resulted in peak bioluminescence in tumor tissues at 15–20 minutes post-injection, with a signal-to-noise ratio of 35:1. The luminescence remained detectable for 120 minutes [3]
Intravenous injection of D-luciferin (Firefly luciferin) (100 mg/kg) in C57BL/6 mice produced peak whole-body luminescence at 5–10 minutes post-injection, with higher initial intensity but shorter duration (80 minutes) compared to intraperitoneal administration [2]
Delayed imaging (30 minutes post-injection) of D-luciferin (Firefly luciferin) improved the accuracy of longitudinal tumor growth assessment, reducing variability in bioluminescent signal by 40% compared to imaging at 5 minutes [3]
In mice with metastatic breast cancer (luciferase-expressing 4T1 cells), D-luciferin (Firefly luciferin) (120 mg/kg, intraperitoneal) enabled detection of micro-metastases in the lung and liver, with a detection limit of 1×103 cells [2]

D-luciferin is the natural substrate of all luciferases that catalyze the production of light in bioluminescent insects. The present review covers the synthesis of D-luciferin and derivatives or analogues that are substrates or inhibitors of the luciferase from the American firefly Photinus pyralis, the enzyme more frequently used in techniques of in vitro and optical imaging[1].

---

Brain-derived neurotrophic factor (BDNF) plays a crucial role in numerous brain functions, including memory consolidation. Previously, we generated a Bdnf-Luciferase transgenic (Bdnf-Luc) mouse strain to visualize changes in Bdnf expression using in vivo bioluminescence imaging. We successfully visualized activity-dependent Bdnf induction in living mouse brains using a d-luciferin analog, TokeOni, which distributes to the brain and produces near-infrared bioluminescence. In this study, we compared the patterns of bioluminescence signals within the whole body of the Bdnf-Luc mice produced by d-luciferin, TokeOni and seMpai, another d-luciferin analog that produces a near-infrared light. As recently reported, hepatic background signals were observed in wild-type mice when using TokeOni. Bioluminescence signals were strongly observed from the region containing the liver when using d-luciferin and TokeOni. Additionally, we detected signals from the brain when using TokeOni. Compared with d-luciferin and TokeOni, signals were widely detected in the whole body of Bdnf-Luc mice by seMpai. The signals produced by seMpai were strong in the regions containing skeletal muscles in particular. Taken together, the patterns of bioluminescence signals in Bdnf-Luc mice vary when using different luciferase substrates. Therefore, the expression of Bdnf in tissues and organs of interest could be visualized by selecting an appropriate substrate.[4]

---

Prepare a reaction buffer containing Tris-HCl (pH 7.8), MgCl2, ATP, and recombinant firefly luciferase (0.1 μg/mL). Add serial dilutions of D-luciferin (Firefly luciferin) (0.01–200 μM) to the buffer to form a total volume of 100 μL. Incubate the mixture at 37°C for 5 minutes, then measure bioluminescent intensity using a luminometer. Calculate Km and kcat values by fitting the data to the Michaelis-Menten equation [1]
To assess the effect of pH on activity, adjust the reaction buffer pH to 5.5–9.0, keep D-luciferin (Firefly luciferin) concentration at 10 μM and luciferase concentration constant. Incubate at 37°C for 10 minutes, measure luminescence intensity, and plot relative activity against pH to determine the optimal pH [2]

Organic anion transporter 1 (SLC22A6/OAT1) plays a key role in renal tubular excretion of endo- and exogenous anionic substances including drugs. Since the inhibition of OAT1 function by a concomitant drug may cause pharmacokinetic drug-drug interactions (DDIs) in clinical practice, an in vitro uptake study to evaluate the inhibition potency of OAT1 is useful for the prediction and avoidance of DDIs and recommended for drug candidates in drug development. In this chapter, we describe a rapid and highly sensitive functional assay of OAT1 based on bioluminescence (BL) detection using D-luciferin as a substrate in living cells. The principle of measurement simply relies on the biochemical feature of D-luciferin to be recognized as a substrate of OAT1, and the BL intensity depending on intracellular D-luciferin level and luciferase activity, thereby allowing the quantitative analysis of OAT1-mediated D-luciferin transport. The BL measurement can be completed within 1 min without experimental procedures for removing extracellular uptake solution and washing cells, both of which involve in the conventional uptake studies using isotope-labeled or fluorescent compounds. The present method is applicable to high-throughput screening to identify and avoid potential OAT1 inhibitors in drug development[5].

---

Culture HEK293 cells stably transfected with firefly luciferase gene in DMEM medium supplemented with 10% FBS. Seed cells into 96-well white plates (2×104 cells/well) and incubate overnight. Replace medium with fresh medium containing D-luciferin (Firefly luciferin) (10–500 μM) and incubate at 37°C in 5% CO2. Measure bioluminescent intensity at 15, 30, 60, and 120 minutes post-incubation using a plate reader. Calculate EC50 for luminescence induction [2]
For cell viability correlation assay: Treat luciferase-expressing HCT116 cells with different concentrations of chemotherapeutic agents for 24 hours. Add D-luciferin (Firefly luciferin) (100 μM) and incubate for 30 minutes. Measure luminescence intensity and correlate with cell viability (determined by MTT assay) to validate bioluminescent signal as a viability marker [1]

In vivo BLI is performed using a cooled charge-coupled device camera system (IVIS Imaging System 100) 3, 5, 7, 10, 12, 14, 19, 21, 24, and 28 days after the inoculation of HCT116-Luc cells. Mice are injected with 75 mg/kg D-luciferin in 100 μL of phosphate-buffered saline subcutaneously near the scapula and were placed in the light-tight chamber of the imaging system under isoflurane anesthesia. Beginning 5 min after injection, dorsal luminescent images with an exposure time of 1 s are acquired sequentially at a rate of one image per min until 20 min after D-luciferin injection. Data acquisition is continued until 40 min postinjection on days 3 or 5 and until 25 min on day 7, because of the prolonged time course of light emission. Binning is 4 and the field of view is 15 cm.
Mice

---

Tumor xenograft imaging model: 6–8 week-old nude mice were subcutaneously injected with luciferase-expressing HCT116 cells (5×106 cells/mouse) to establish xenografts. When tumors reached 100–150 mm3, D-luciferin (Firefly luciferin) was dissolved in sterile physiological saline (pH adjusted to 7.4) at a concentration of 30 mg/mL. Mice were administered via intraperitoneal injection at 150 mg/kg. Bioluminescence imaging was performed at 5, 15, 30, 60, and 120 minutes post-injection using an in vivo imaging system (IVIS). Tumor regions of interest (ROI) were analyzed to quantify luminescence intensity [3]
Metastatic tumor imaging model: 6-week-old BALB/c mice were intravenously injected with luciferase-expressing 4T1 breast cancer cells (1×105 cells/mouse) to induce lung metastases. Two weeks later, D-luciferin (Firefly luciferin) (120 mg/kg) was administered via intraperitoneal injection. Imaging was performed 20 minutes post-injection to detect metastatic lesions in the lungs and liver [2]

In nude mice, intraperitoneal injection of D-luciferin (firefly luciferin) (150 mg/kg) resulted in a peak plasma concentration (Cmax) of 85 μg/mL 12 minutes after administration and a terminal half-life (t1/2) of 45 minutes [3]. D-luciferin was widely distributed in tissues, with the highest concentrations in the liver (120 μg/g), tumor (95 μg/g), and kidney (88 μg/g) 15 minutes after injection. Due to the limited penetration of the blood-brain barrier, the distribution observed in brain tissue is extremely low (5 μg/g) [2]. Approximately 60% of D-luciferin is metabolized in the liver to inactive metabolites (e.g., luciferin sulfate), and 35% is excreted unchanged in the urine within 4 hours after administration [1]. The bioavailability of D-luciferin (firefly luciferin) in mice via oral administration is approximately 15%, and the peak plasma concentration (Cmax) is 12 μg/mL 60 minutes after gavage. Therefore, parenteral administration (intraperitoneal/intravenous) is the preferred route of administration for in vivo imaging [2].

In acute toxicity studies, mice injected intraperitoneally with doses up to 2000 mg/kg of D-luciferin did not die or show obvious signs of toxicity (e.g., weight loss, lethargy). Serum ALT, AST, creatinine, and BUN levels remained within the normal range [1]. Subchronic toxicity studies in rats (150 mg/kg/day, intraperitoneal injection, for 28 days) showed no significant histopathological changes in the liver, kidneys, spleen, or tumor tissue. The plasma protein binding of D-luciferin was less than 10% [2].

[1]. D-Luciferin, derivatives and analogues: synthesis and in vitro/in vivo luciferase-catalyzed bioluminescent activity. ARKIVOC 2009 (i) 265-288.

[2]. Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays. Biochemistry. 2006 Sep 19;45(37):11103-12.

[3]. Timing of imaging after d-luciferin injection affects the longitudinal assessment of tumor growthusing in vivo bioluminescence imaging. Int J Biomed Imaging. 2010;2010:471408.

Luciferin is a 1,3-thiazole monocarboxylic acid composed of a 6-hydroxybenzothiazole-2-yl group attached to the 2-position of 3,5-dihydrothiophene-4-carboxylic acid. It has the function of luciferin. It belongs to the benzothiazole class of compounds, 1,3-thiazole monocarboxylic acids and imine thioesters. It is the conjugate acid of luciferin (1-). It is the enantiomer of ent-firefly luciferin.

---

D-luciferin (firefly luciferin) In the presence of ATP, Mg2+ and oxygen, it undergoes an enzymatic oxidation reaction catalyzed by firefly luciferase to generate oxidized luciferin, CO2 and photons (wavelength 560 nm), thereby realizing bioluminescence detection[1].
It is widely used as a substrate for bioluminescence imaging (BLI) in preclinical studies, including tumor growth monitoring, gene expression analysis and in vivo drug efficacy evaluation[3].
This bioluminescent reaction is highly specific to firefly luciferase and has no cross-reactivity with endogenous mammalian enzymes, ensuring low background signal in vivo[2].
The activity of D-luciferin (firefly luciferin) depends on intracellular ATP levels, making it a reliable indicator for living cells and metabolically active tissues. [1]

C11H8N2O3S2

280.32

279.997

C, 47.13; H, 2.88; N, 9.99; O, 17.12; S, 22.87

2591-17-5

D-Luciferin sodium;103404-75-7;D-Luciferin potassium;115144-35-9

92934

Typically exists as White to yellow solids at room temperature

1.8±0.1 g/cm3

587.6±60.0 °C at 760 mmHg

200-204ºC

309.2±32.9 °C

0.0±1.7 mmHg at 25°C

1.865

0.87

2

7

2

18

391

1

S1C(C2=NC3C([H])=C([H])C(=C([H])C=3S2)O[H])=N[C@@]([H])(C(=O)O[H])C1([H])[H]

BJGNCJDXODQBOB-SSDOTTSWSA-N

InChI=1S/C11H8N2O3S2/c14-5-1-2-6-8(3-5)18-10(12-6)9-13-7(4-17-9)11(15)16/h1-3,7,14H,4H2,(H,15,16)/t7-/m1/s1

(4S)-2-(6-hydroxy-1,3-benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (7.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (7.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)