1. Notes a) D-luciferin dissolves readily in aqueous buffer with a pH of 6.1–6.5, with a maximum solubility of 100 mM. It is possible to produce the stock solution with ATP-free water and keep it in the dark at -20°C. To dissolve, free acids need to be neutralized with the proper base. Fluorescein will racemize to the L-isomer and generate dehydrofluorescein under base catalysis at higher pH levels. b) D-luciferin is compatible with all current ATP assay systems including reporter assays. /> c) To reduce the possibility of ATP contamination, use hands when testing for ATP and use ATP-free containers. Use only reagents and sterilized, ATP-free water. For all reagent preparation, use autoclaved water. 2. Experimental Protocol: This protocol should be adjusted to meet your unique requirements, as it simply serves as a reference. An illustration of how potassium and potassium are prepared with sodium salts is shown in the scheme below. It works well for in vivo animal application and the majority of cell types. 2.1 An example of a process for analyzing bioluminescence images in vitro a) Make a stock solution of 100 mM (100-200X) fluorescein in sterile water. Blend thoroughly. Use right away, or repack all at once; store at -20°C; keep out of direct sunlight and avoid frequent freezing and thawing. b) Heat the tissue culture medium and prepare the working solution for D-Luciferin (0.5–1 mM). c) Take the culture medium out of the cells that have been cultivated. d) Before imaging, add the luciferin working solution to the cells and incubate them for five to ten minutes at 37°C. 2.2 An example in vivo bioluminescence image analysis technique is as follows: a) Make a 15 mg/mL stock solution of fluorescein in DPBS free of Mg2+ and Ca2+. well combined. c) Sterilize the mixture by passing it through a 0.2 μM filter. Use right away, or aliquot for one-time use; store at -20°C; keep out of the freezer and thaw cycles; keep out of direct sunlight. c) Ten to fifteen minutes prior to imaging, inject luciferin 150 mg/kg (or 10 μL/g of luciferin stock solution) intraperitoneally (ip). NOTE: To ascertain the peak signal time, luciferin kinetic investigations ought to be carried out for every animal model. 2.3 An example of a technique for detecting luciferin reporter genes: a) Make a stock solution of 100 mM luciferin in sterile water. Use right away, or repack all at once; store at -20°C; keep out of direct sunlight and avoid frequent freezing and thawing. b) Prepare a 25 mM tricine buffer pH 7.8 solution containing 1 mM D-Luciferin working solution, 3 mM ATP, 1 mM DTT, and 15 mM MgSO4. d) Fill the microplate with 5 to 10 μL of cell lysate. As a blank, use lysis reagent or buffer devoid of lysate. d) As directed by the manufacturer, prime the luminometer using fluorescein working solution. e) Inject 200 μL of the fluorescein working solution right away; allow 10 seconds for integration.

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.

[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

C11H8N2O3S2

280.32

2591-17-5

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

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

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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.

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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.

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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.

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