1. The D-luciferin salt exhibits a high degree of solubility, up to 100 mM, in aqueous buffer (pH 6.1-6.5). The water component of ATP is present in the volatile liquid, which is kept at -20°C in the dark. A suitable alkaline neutralizing solution must be used to dissolve the free acid. Under base catalysis, fluorescein will form dehydrofluorescein at the pH value mentioned above and racemize to L-eleven. b) Any current reporter assay or ATP assay system can be utilized with D-luciferin. c) To reduce the possibility of ATP contamination, if testing ATP, use clean ATP containers and wear gloves. Use reagents and water that are sterile and free of ATP. To prepare all of the reagents, use autoclaved water. 2. Experimental protocol: This protocol should be adjusted to meet your unique requirements, as it only serves as a guide. An example of a sodium salt preparation of potassium and potassium is shown in the following scheme. Large 2.1 In vitro protocol Example of Bioluminescence Image Analysis batches can benefit from it. a) Make a stock solution of 100 mM (100-200X) fluorescein in sterile water. Stir thoroughly. Prepare a 0.5–1 mM D-luciferin working solution in tissue cells that have been preheated. Use right away, or dispense once. Temperature: -20°C. Aspirate the cells out of the cell (c). a) Prepare a 15 mg/mL fluorescein stock solution in DPBS to clear Mg2+ and Ca2+. d) Add cultured fluorescent agent to cells. 2.2 Sample protocol for internal bioluminescence image analysis. b) Filter: Run a 0.2 μM filter through the solution to sterilize it. Avoid freeze-thaw cycles, light exposure, and use right away, or aliquot and store at -20°C. c) Ten to fifteen minutes prior to imaging, administer luciferin intraperitoneally (ip) at a dose of 150 mg/kg (or 10 μL/g of luciferin stock solution) to the animal. Note: Every animal model should be used for luciferin kinetic investigations. Example luciferin reporter gene detection protocol (high-frequency signal time 2.3) a) Make a 100 mM luciferin stock solution in sterile water. Use right away, or aliquot once; store at -20°C; keep out of the freezer and thaw cycles; keep out of direct sunlight. Create a 1 mM D-Luciferin working solution in 25 mM Tricine buffer pH 7.8 by adding 3 mM ATP, 1 mM DTT, and 15 mM MgSO4. c) Spoon 5–10 μL of the cell transporter into the plate. Use d) In accordance with the manufacturer's instructions, prime the luminometer using fluorescein working solution. e) Inject 200 μL of the working fluorescein solution right away; the integration time is 10 s.

The most popular method at the moment is bioluminescence (BLI), which uses D-luciferin substrate and firefly luciferase (Fluc) as a reporter gene. A time-intensity curve was created by graphing the overall signal intensity versus the amount of time following D-luciferin injection. Apart from the peak signal, surrogate signals for the peak signal were identified as the signals at predetermined 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]. Use 10 μL of D-luciferin stock solution (intraperitoneal or intravenous) for every gram of body weight. An injection of 20 g should typically contain 200 μL due to the conventional dose of 150 mg/kg. To dissolve the D-luciferin (potassium or sodium salt) solution to a final concentration of 15 mg/mL, thaw it and dilute it in dPBS (clear calcium or magnesium). Wet a 0.22 µM filter with 5–10 mL of sterile HO, then drain. ..Pass the D-luciferin solution through a 0.22 µM syringe filter that has been produced.

[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

C11H7N2NAO3S2

302.29

301.9796

103404-75-7

D-Luciferin;2591-17-5;D-Luciferin potassium;115144-35-9

Typically exists as solids (or liquids in special cases) at room temperature

O=C([C@@H]1N/C(SC1)=C(N=C2C=C3)\SC2=CC3=O)[O-].[Na+]

BZNVUYVALNTPBG-WJCSTRGMSA-M

InChI=1S/C11H8N2O3S2.Na/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

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

D-Luciferin Sodium

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 : ~250 mg/mL (~826.99 mM)
DMSO : ~100 mg/mL (~330.80 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.27 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.27 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 25.0 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.

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Solubility in Formulation 3: 100 mg/mL (330.80 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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