Natural substrate of luciferase (Luc) enzyme

1. Note:
a) The D-luciferin salts (sodium or potassium) exhibits a high degree of solubility, up to 100 mM, in aqueous buffer (pH 6.1-6.5). The stock solution can be prepared with ATP free water and stored in the dark at -20 ° C. Free acids must be neutralized with an appropriate base in order to dissolve. At higher pH values, fluorescein will form dehydrofluorescein under alkaline catalysis and racemize into L-isomer (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.
The following scheme is an example of preparation of D-Luciferin sodium/potassium salts. It is suitable for most cell types and in vivo animal use.
2.1 Example of In vitro Bioluminescence Image Analysis.
a) Prepare 100 mM (100-200X) D-fluorescein sodium or potassium stock solution in sterile water. Mix well. Immediately use or aliquot it, store at -20 ° C, avoid freeze-thaw cycles, and avoid exposure to light.
b) Prepare 0.5-1 mM D-Luciferin working solution in pre-heated 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 ° C for 5-10 minutes before imaging.
2.2 Example for analyzing bioluminescence images in vivo
a) Prepare 15 mg/mL D-Luciferin stock solution in DPBS, free of Mg2+and Ca2+. Mix evenly.
b) The filter removes bacteria from the solution through a 0.2 μ M filter. Immediately use or aliquot it and store at -20 ° C to avoid freeze-thaw cycles and exposure to light.
c) 10-15 minutes before imaging, intraperitoneal (IP) injection of D-Luciferin at 150 mg/kg (or 10 μ L/g fluorescein stock solution) of animal body weight.
Attention: Fluorescein kinetics studies should be conducted on each animal model to determine peak signal time.
2.3 Example for D-Luciferin Reporter Gene Testing
a) Prepare 100 mM D-Luciferin stock solution in sterile water. Immediately use or aliquot it, store at -20 ° C, avoid freeze-thaw cycles, and avoid exposure to light.
b) Prepare 1 mM D-Luciferin working solution and 3 mM ATP, 1 mM DTT, and 15 mM MgSO4 in 25 mM Tricine buffer 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-Luciferinworking solution.
e) Immediately inject 200 μ L of D-Luciferin working solution, with an integration time of 10 seconds.

The most popular technology 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]. For each gram of body weight, inject 10 μL of D-luciferin (i.p. or intravenous) stock solution; a conventional dosage of 150 mg/kg for a 20 g mouse would be roughly 200 μL. 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. Pipette 5–10 mL of sterile water to prewet the 0.22 μm filter; discard the water. Filter the D-luciferin solution using a prepared 0.22 µm syringe filter to ensure it is sterile.[3]

---

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]

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].

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].

The peak signal or the signal at a predetermined, fixed time point after D-luciferin injection may be used for the quantitative analysis of in vivo bioluminescence imaging. We repeatedly performed sequential bioluminescence imaging after subcutaneous injection of D-luciferin in mice bearing subcutaneous tumors. The peak time in each measurement became shorter early after cell inoculation, presumably due to gradual establishment of intratumoral vasculature, and reached a plateau of about 10 min on day 10. Although the correlation between the signal at a fixed time point and the peak signal was high, the signal at 5 or 10 min normalized for the peak signal was lower for earlier days, which caused overestimation of tumor growth. The time course of the signals after D-luciferin injection may vary with time after cell inoculation, and this variation should be considered when determining the imaging protocol for quantitative bioluminescence tumor monitoring.[2]

[1]. Giuseppe Meroni, et al. D-Luciferin, derivatives and analogues: synthesis and in vitro/in vivo luciferase-catalyzed bioluminescent activity. ARKIVOC 2009 (i) 265-288.
[2]. Rajesh Shinde, et al. Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays. Biochemistry. 2006 Sep 19;45(37):11103-12.
[3]. Inoue Y, et al. 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.
[4]. Bioluminescence imaging using d-luciferin and its analogs for visualizing Bdnf expression in living mice; different patterns of bioluminescence signals using distinct luciferase substrates. J Biochem 2022 Oct 19;172(5):321-327.
[5]. A Simple and Rapid Bioluminescence-Based Functional Assay of Organic Anion Transporter 1 as a D-Luciferin Transporter. Methods Mol Biol 2022:2524:119-126.

Staining Example 1:
D-Luciferin potassium may be used as a substrate of luciferases for in vivo imaging.
Method: For bioluminescence imaging.
1). Anesthetize mice, then inject mice with D-Luciferin potassium (75 mg/kg) for image.
2). Use the bioluminescence imaging system for image.

---

Staining Example 2:
D-Luciferin potassium may be used as a substrate of luciferases for in vivo imaging to monitor tumor growth.
Method: For bioluminescence imaging.
1). Inject D-Luciferin potassium (150 mg/kg; intraperitoneal injection) into the mice.
2). Use a bioluminescence imaging system for image.

---

Staining Example 3:
D-Luciferin potassium may be used as a substrate of luciferases for a split-luciferase (LUC) assay.
Method: For split-luciferase (LUC) assay.
1). Incubate plant sample (leaves) with D-Luciferin potassium (1 mM; 10 min) luciferin.
2). Use a Photek camera to capture signals and images.

---

Staining Example 4:
D-Luciferin may be used as a substrate of luciferases for in vivo imaging to monitor tumor growth.
Method: For bioluminescence imaging.
1). Inject D-Luciferin potassium (150 mg/kg; intraperitoneal injection) into the mice.
2). Use IVIS Lumina XRMS Series for bioluminescence imaging.

C11H7KN2O3S2

318.4

317.9535

C, 41.49; H, 2.22; K, 12.28; N, 8.80; O, 15.07; S, 20.14

115144-35-9

D-Luciferin sodium;103404-75-7;D-Luciferin;2591-17-5; 103404-75-7 (Sodium)

135478035

Typically exists as Light yellow to yellow solids at room temperature

1.384

1

7

2

19

396

1

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

PWQWXGFOCJCDIF-SREJTOIWSA-M

InChI=1S/C11H8N2O3S2.K/c14-5-1-2-6-8(3-5)18-10(12-6)9-13-7(4-17-9)11(15)16/h1-3,7,13H,4H2,(H,15,16)/q+1/p-1/b10-9-/t7-/m1./s1

potassium (S,Z)-2-(6-oxobenzo[d]thiazol-2(6H)-ylidene)thiazolidine-4-carboxylate

D-Luciferin potassium salt; Potassium (S)-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate; D-Luciferin (potassium); D-Luciferin (potassium salt); 4-Thiazolecarboxylic acid, 4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)-, potassium salt (1:1), (4S)-; (S)-2-(6-Hydroxy-2-benzothiazolyl)-2-thiazoline-4-carboxylic acid potassium salt; D-Luciferin,potassium salt; D-Luciferin potassium

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ~25 mg/mL (~78.52 mM)

Solubility in Formulation 1: 8.33 mg/mL (26.16 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

---

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