1. Precautions prior to operation: a) D-luciferin dissolves readily in an aqueous buffer at a concentration of 100 mM (pH 6.1–6.5). Dissolution solutions can be prepared in transparent ATP water and kept under light at -20°C. To dissolve the free acid, the right base needs to be used to neutralize it. Fluorescein is produced by racemization to the L-hydroxy form and base-catalyzed deoxyradical formation at the pH values mentioned above. b) Any current reporter gene 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. 2. Experimental Operations: This instruction only serves as a reference for experiments; the precise techniques used in each experiment will be modified to suit your needs. 2.1 In vitro experimental simulation example a) Make a stock solution of 100 mM (100-200X) luciferin in sterile water, then mix it thoroughly. Use right away or divide into aliquots for single use, and store at -20°C to prevent freezing and thawing as well as exposure to sunlight. b) Mix the pre-cultured tissue fluid with 0.5–1 mM D-Luciferin working solution. c) Take the bone marrow out of each cell. d) Before imaging, give the cells a shot of D-Luciferin working solution and let them sit at 37°C for five to ten minutes. 2.2 In vivo Imaging Experiment Example a) Prepare a 15 mg/mL stock solution of fluorescein in DPBS and evenly mix transparent Mg2+ and Ca2+. b) Sterilize the mixture by passing it through a 0.2 μm filter. Use right away, or take aliquots and incubate at -20°C all at once. c) Give animals an intraperitoneal injection of D-luciferin 10 μL/g of stock solution or 150 mg/kg of the drug 10–15 minutes prior to inference. .2.3 Gene detection experiment example a) Make a stock solution of 100 mM luciferin in sterile water. To prevent freeze-thaw cycles and sun damage, use right away or use aliquots at a time and store at -20°C. b) Make 1 mM D-Luciferin in 25 mM tricine buffer (pH 7.8) with 3 mM ATP, 1 mM DTT, and 15 mM MgSO4. c) Fill a microplate with 5–10 μL of cell mitochondrial fluid using a pipette. inside the aperture plate. As blank controls, use buffers or precursor reagents. d) For photometry, use the fluorescein working solution as directed. e) Inject 200 μL of the working solution of D-Luciferin right away, giving it ten seconds to bind.

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.

[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

C11H7KN2O3S2

318.4

317.9535

115144-35-9

D-Luciferin sodium;103404-75-7;D-Luciferin;2591-17-5

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

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

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)

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).

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