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Firefly Luciferin

Content and storage 

Material Amount Storage Stability
Firefly Luciferin 1 g
  • -20 °C
  • Desiccate
  • Protect from light
When stored as directed, reactive probes are stable for at least 6 months
5 g
1 g is enough for 300 mice

General Specifications 

Color:  Light yellow powder 

Detection Method: Bioluminescence

Excitation Class: Visible 

Molecular Weight: 280.32 g.mol-1

Formula: C11H8N2O3S2

Cas Number: 2591-17-5 

Shipping Condition: Dry Ice 

Regulatory Statement: For Research Use Only. Not for use in diagnostic procedures. 

Description

Luciferin is a small molecule that consists of a benzothiazole moiety attached to a thiazole carboxylic acid moiety. Luciferin is a natural substrate of luciferase, an enzyme found in Firefly luciferase. This molecule has fluorescent properties (Ex 328 nm; Em 532 nm in H2O)1,2 but it is mainly used for bioluminescent imaging (BLI) purpose.

Bioluminescence is a natural process that has been found in various living organisms such as the North American firefly (Photinus pyralis) and is based on the oxidation of D-luciferin catalyzed by the enzyme Luciferase. Upon recognition by the enzyme, D-luciferin is oxidized to oxy-luciferin and releasing one photon of light, which can be detected by a CCD camera (Figure 1). The intensity of the light output is closely related to the amount of D-luciferin available for the enzyme and therefore it is possible to quantify the amount of luciferin by measuring the amount of emitted light with a CCD camera. As a non-invasive imaging method, BLI is comparable to other in vitro and in vivo techniques but has the advantage of high sensitivity, convenience and ease of use. It does not require a light source (as opposed to fluorescence) and there is no background signal from tissues that do not express luciferase. Moreover, it allows real-time imaging of luciferase expressing cells or luciferase expressing mice.3-5 The luciferin/Luciferase process is very substrate dependent and does not allow significant modification on the luciferin scaffold to be recognized by the enzyme.

Figure 1: Oxydation of D-luciferin by Firefly luciferase forming oxy-luciferin and a photon of light

Guideline for use 

D-luciferin for in vitro use:

- 1g of D-luciferin is dissolved in 33.3 mL of sterile water to make a 30 mg/mL stock solution. After mixing and filtering (0.2 um), the solution is aliquoted and purged with nitrogen (inert gas prevents oxidation) protect from light and freeze down to -80°C for future use. Frozen stock solutions can be stored up to 1 year.

- D-luciferin stock solution is thawed, kept on ice and protected from light. Stock solution is diluted at 1:200 in complete culture medium to 150 ug/mL. Luciferase expressing cells are incubated 5 min at 37°C prior to imaging. Diluted solution should be discarded after use.

D-luciferin for in vivo use:

- 1g of D-luciferin is dissolved in 66.6 mL of DPBS, w/o Mg2+ and Ca2+ to make a 15mg/mL solution. After mixing and filtering, the solution is aliquoted and purged with nitrogen (inert gas prevents oxidation) protect from light and freeze down to -80°C for future use. Frozen stock solution can be stored up to 1 year.

- D-luciferin solution is thawed, kept on ice and protected from light. Diluted solution should be discarded after use

- Mouse is injected IP with D-luciferin solution at 150mg/kg of body weight. Image are acquired after 10-15 min post-injection.

References: 

1. White, E.H. et al.,(1963) J. Am. Chem. Soc., 85, 337

2. Bowie, L.J., (1978) Methods in Enzymology, 57, 23

3. Prescher, J. A., and Contag, C. H. (2010) Curr. Opin. Chem. Biol. 14, 80-89.

4. McCaffrey, A., Kay, M. A., and Contag, C. H. (2003) Mol. Imaging 2, 75-86

5. Massoud, T. F., and Gambhir, S. S. (2003) Genes Dev. 17, 545-580. 

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