Cell lines

B16-F10 and MCF-7 clonal cells

Preparation Method

Cells that stably express luciferase were seeded in 12-well plates (2 × 10³/well). Cells were treated with D-luciferin substrate diluted in media or media alone.

Reaction Conditions

30 min incubation at 37℃ in the dark, washed with PBS once and replaced the normal growth medium.

Applications

D-luciferin could be used to investigate whether biophotonic activity or the luciferase gene itself have negative influence on cell growth in vitro. Luciferase bioluminescence was not sufficient to generate photodynamic toxicity in vitro. It has little difference of cell viability between clones expressing different levels of luciferase or between cells periodically treated with D-luciferin.

Animal models

BALB/C nude (females), aged 6–8 weeks

Preparation Method

HEK 293T cells were prepared at a concentration of 2 × 10⁶cells/ml in PBS; Injecting 1 × 10⁵HEK 293T cells into both flanks of mice immediately, images were acquired after injection of D-luciferin.

Dosage form

150 mg/kg

Applications

The BLI measurements were performed when the peak of emission occurred, after injection of the luciferins into animals. Luc2/ D-luciferin, CBG99/ D-luciferin, CBR2/ D-luciferin, and Akaluc/ D-luciferin produced peaks at 610 nm, 540 nm, 620 nm, and 640 nm, respectively. When CycLuc1 was used as a substrate, the emission peak of Luc2 and Akaluc were green shifted towards 600 nm. Analysed the total emission of each luciferase in vivo with D-Luciferin or the luciferin analogues, Luc2, CBG99, and CBR2 paired with D-luciferin produced the highest signals which were 20-fold higher than that of Akaluc/ D-luciferin (p value < 0.001).

D-Luciferin sodium salt is the substrate of luciferases that catalyze the production of light in bioluminescent insects.

D-Luciferin is a common substrate for luciferase and is frequently used in the entire biotechnology field, especially in vivo imaging techniques. The mechanism of action of the imaging is that D-luciferin (substrate) is oxidized to emit light under the action of ATP and luciferase. When D-luciferin is in excess, the number of photons produced is positively correlated with the concentration of luciferase. After transfecting a plasmid carrying a luciferase-encoding gene (Luc) into cells, cells with Luc is engrafted into research animals, and then injected with D-luciferin to detect changes in light intensity through bioluminescence imaging (BLI). The effect of ATP on this reaction system can also be used to indicate energy or vital signs based on changes in bioluminescence intensity.^[1][2]

D-luciferin reacts with luciferase, ATP and oxygen with light emission, and the light is detected by a sensitive photographic film.^[3]All in vitro D-luciferin measurements were obtained after 1 min at 37 ℃ using a 30-s acquisition time using a series of filters ranging from 520 to 800 nm. In vitro analysis demonstrated that at a relatively low, but biologically relevant (in vivo) substrate concentration (0.1 mM), three of the four luciferases give maximum signal when combined with D-luciferin.^[4]

In vivo analysis showed that the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was D-luciferin. CBR2 gave the brightest signal with the near-infrared substrate, NH2-NpLH2. When paired with CycLuc1 or Akalumine-HCl, Akaluc was brighter. Besides, new combinations of luciferases with distinct colors having potential for multiplexing with a single substrate in superficial tissue were recommended, such as CBG99/ D-luciferin (540 nm) and CBR2/ D-luciferin (620 nm); CBG99/ D-luciferin (540 nm) and Luc2/ D-luciferin (610 nm).^[4]

References:
[1] McElroy WD. The Energy Source for Bioluminescence in an Isolated System. Proc Natl Acad Sci U S A. 1947 Nov;33(11):342-5. doi: 10.1073/pnas.33.11.342.
[2] GREEN A, MCELROY WD. Function of adenosine triphosphate in the activation of luciferin. Arch Biochem Biophys. 1956 Oct;64(2):257-71. doi: 10.1016/0003-9861(56)90268-5.
[3] Hauber R. A New, Very Sensitive, Bioluminescence-Enhanced Detection System for Protein Blotting. J. Clin. Chem. Clin. Biochem. 1987,25, pp. 511-514. doi:10.1515/cclm.1987.25.8.511.
[4] Zambito G. Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging. Mol Imaging Biol. 2020 Dec;22(6):1523-1531. doi: 10.1007/s11307-020-01523-7.