Optical in vivo imaging technology currently primarily utilizes two techniques: bioluminescence and fluorescence. Bioluminescence relies on the principle that luciferase can catalyze the chemical luminescence of substrates (D-Luciferin or Coelenterazine). Cells stably expressing luciferase are implanted into animals, and upon subsequent injection of substrates into the body, a reaction occurs. The optical system detects the intensity of light, indirectly reflecting changes in cell number or cell localization. This technology has been widely applied in various fields, including the establishment of tumor or disease animal models, as well as in virology research, siRNA research, stem cell research, and protein interaction studies.
Principle
There are two common types of luciferases: firefly luciferase (encoded by the luc gene) and Renilla luciferase (encoded by the Rluc gene). The substrate for firefly luciferase is D-Luciferin, while the substrate for Renilla luciferase is Coelenterazine. The common principle of their action is that, in the presence of ATP and luciferase, the substrate undergoes oxidation and emits light (with different substrates emitting light of different colors and wavelengths). When the substrate is in excess, the number of produced photons is positively correlated with the concentration of luciferase.
D-Luciferin
D-Luciferin: There are three types, namely D-Luciferin, Sodium Salt/D-luciferin sodium salt, D-Luciferin, Potassium Salt/D-luciferin potassium salt, and D-Luciferin Firefly, free acid/D-firefly luciferin. Arcegen offers several commonly used types of D-Luciferin, each batch of reagent undergoes strict quality validation (monitored by 9 independent HPLC and FTIR standard methods) and luminescence efficiency testing, ensuring high repeatability and success rate for each experiment. All products can be used for in vivo imaging analysis and in vitro chemiluminescence detection experiments. Arcegen's D-Luciferin is a synthetic firefly luciferin, with a structure identical to the beetle luciferin provided by Promega, and is a water-soluble substrate for firefly luciferase.
（1）D-Luciferin, Sodium Salt
Molecular formula: NaC11H7N2O3S2·H2O Molecular weight: 320.32 g/mol Purity: High purity (99.7%)
Applications: 1) In vitro chemiluminescence analysis; 2) In vivo imaging experiments; 3) High sensitivity ATP analysis;
Procedure:
Protocol 1：In Vitro Bioluminescent Assays
1) Dissolve 1.0 g of D-luciferin sodium salt in 33.3 mL of distilled water to prepare a 100 mM stock solution (200×, concentration 30 mg/ml). Use immediately after thorough mixing or aliquot and store at -20°C.
2) Dilute the stock solution with tissue culture medium at a ratio of 1:200 to prepare a working solution (final concentration 150 μg/mL).
3) Remove the culture medium from the cultured cells.
4) Prior to imaging analysis, add 1× luciferin working solution to the cells and proceed with imaging analysis.
Protocol 2：In vivo analysis
1) Prepare a working solution of D-luciferin sodium salt (15 mg/mL) using sterile PBS (w/o Mg2+, Ca2+), and filter sterilize through a 0.2 μm filter membrane. Keep the solution cold and protected from light once used.
2) The injection volume depends on the injection method, as follows:
|
Injection methods |
Dosage |
|
Intravenous injection (25-27 gauge needle) |
Add the corresponding volume of 15 mg/mL luciferin working solution per 10 μL/g body weight concentration. |
|
Intraperitoneal injection (25-27 gauge needle) |
Add the corresponding volume of 15 mg/mL luciferin working solution per 10 μL/g body weight concentration. |
|
Intramuscular injection (27 gauge needle) |
50 μL of 1–2 mg/mL luciferin working solution. |
|
Intranasal injection (pipette) |
50 μL of 3 mg/mL luciferin working solution. |
3) Imaging analysis should be conducted 5-10 minutes after injection into the body.
（2）D-Luciferin, Potassium Salt
Molecular formula: NaC11H7N2O3S2·H2O Molecular weight: 320.32 g/mol Purity: High purity (99.7%)
Applications:
1) Imaging analysis of luciferase-labeled genes and luciferase-fusion genes expressed in live cells, tissues, or organisms, both in vivo and in vitro.
2) Widely used in reporter gene analysis, immunological analysis, and ATP fluorescence health monitoring analysis.
Protocol 1: In Vitro Bioluminescent Assays
1) Prepare a 100 mM stock solution (200×, concentration 30 mg/ml). Mix well and use immediately or aliquot and store at -20°C.
2) Dilute the stock solution with pre-warmed tissue culture medium at a ratio of 1:200 to prepare the working solution (final concentration 150 μg/mL).
3) Remove the culture medium from the cells until no residual medium remains.
4) Immediately add 1× luciferin working solution to the cells before imaging analysis, then proceed with imaging analysis (or incubate cells at 37°C for a short time before detection to enhance the signal).
Protocol 2: In vivo analysis in mice
1) Prepare a solution of D-luciferin potassium salt (15 mg/mL) using sterile DPBS (w/o Mg2+, Ca2+), filtered through a 0.2 µm membrane for sterilization. Keep cold and protected from light once prepared.
2) Inject the appropriate volume of 15 mg/mL luciferin solution per gram of body weight into each mouse.
3) Perform imaging analysis 10-15 minutes after intraperitoneal injection. (Conduct luciferin pharmacokinetic studies for each animal model to determine peak signal acquisition time.)
（3）D-Luciferin Firefly,free acid
Molecular formula: C11H8N2O3S2
Molecular weight: 280.330 g/mol
Appearance: White to light yellow powder
Solubility: Soluble at 0.25% concentration in methanol, forming a nearly colorless to light yellow solution
Storage: -15°C, protected from light
Applications:
1) Imaging analysis of luciferase-labeled genes and luciferase-fusion genes expressed in live cells, tissues, or organisms, both in vivo and in vitro.
2) Widely used in reporter gene analysis, immunological analysis, and ATP fluorescence health monitoring analysis.
Example of Application
Treatment effect of in vivo imaging detection of CAR-MUC1 T/CAR-MUC1-IL22 T cells on subcutaneous injection of HN4 cells in mice (Mei Z et al. Cancer Med. 2020)
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