Is it necessary to use a fluorescence reader to measure the luciferase activity in the dual-luciferase reporter gene assay system?

Yes, a fluorescence reader should be used for DLR testing, as the sensitivity of a luminometer is too low, making it difficult to obtain reproducible results.

How do animals respond to repeated injections of D-luciferin?

D-luciferin does not affect animals (there are no significant toxic effects or immune responses).

How stable is the expression of luciferase?

Luciferase genes can be integrated into the cell chromosomes through processes such as stable selection, allowing for continuous and stable expression as the cells divide, migrate, and differentiate. The half-life of luciferase is about 3 hours, so only living cells can continuously express luciferase.

What assays is D-luciferin suitable for?

As a substrate for Firefly luciferase, D-luciferin is commonly used in the following assays: (1) In vivo imaging: Firefly luciferase plays a crucial role in in vivo imaging techniques. By labeling this enzyme to specific cells (such as tumor cells or stem cells) or pathogens, researchers can observe the behavior of these cells or pathogens in living organisms in real-time and non-invasively. (2) In vitro studies: It can be used to analyze ATP levels, assess reporter gene expression, and for various applications such as high-throughput screening and contamination detection.

What is the difference between Beetle luciferin and Firefly luciferin?

There is no difference between the two; it is merely a naming difference by different companies, and their chemical structures are identical.

How is D-luciferin distributed in vivo?

After D-luciferin is injected into the animal, it distributes quickly and easily disperses throughout the body.

What is the in vivo luminescence kinetics of D-luciferin?

D-luciferin can be administered in various ways, with common methods including intraperitoneal injection, subcutaneous injection, and intravenous injection. The kinetic curves vary with different administration routes. For most studies, intraperitoneal/subcutaneous injection reaches peak luminescent signal approximately 10-20 minutes after administration, while intravenous injection reaches peak signal in about 2-5 minutes.

How should the prepared D-luciferin potassium salt solution be stored?

The working solution should be ready for use. Ideally, the prepared storage solution can be stored at -20°C for a short period. If necessary, the luciferin solution can be stored at -20°C for up to 3 weeks. Tips: Storing the above solution at any temperature for an extended period may lead to signal degradation!

What are the forms of luciferin/luciferase substrates?

They are classified into D-luciferin free acid, D-luciferin potassium salt, and D-luciferin sodium salt. Due to their high water solubility, potassium salt and sodium salt are the two most commonly used detection substrates, with potassium salt being the primary form used for in vivo animal studies.

How to determine the peak signal time for different models in small animal in vivo imaging experiments?

The kinetics of bioluminescent signals are tissue-dependent. It is recommended to perform imaging on animals every 5-10 minutes, up to a maximum of 40 minutes, to create a kinetic curve for each new model. (Common peak signal time points are around 10-15 minutes, and luminescence from the luciferin reaction generally starts to decay after 30 minutes and completely disappears after 3 hours.)

Can luciferin substrates cross the blood-brain barrier?

Luciferin substrates are approximately 280 Da, and both their water solubility and lipid solubility are very good, allowing them to easily penetrate cell membranes and the blood-brain barrier.

Advantages of VKEY-BIO’s D-luciferin potassium salt

(1) High purity (≥99%) (2) Excellent bioluminescence performance (3) High solubility (aqueous solution at room temperature >40 mg/ml)

Reaction principle of D-luciferin potassium salt

D-luciferin is a commonly used substrate for luciferase, reacting with luciferase in the presence of Mg²⁺ and ATP, resulting in an oxidative decarboxylation reaction that produces luminescence. Under conditions of excess luciferin, the number of photons produced is positively correlated with the concentration of the enzyme. This type of luminescent reaction can be used for assays such as promoter optimization, drug screening, analysis of cellular ATP levels, and in vivo imaging in animals.

What is the purity of D-luciferin potassium salt? Does the level of purity affect the assay?

The purity of D-luciferin potassium salt is ≥99%. The higher the purity of luciferin, the lower the content of corresponding contaminants; conversely, if the purity is lower, the contaminant concentration will be higher. Excessive levels of contaminants can inhibit certain enzymes and biological processes in cells, potentially leading to deviations in assay results and may cause harm to laboratory animals.

What is the solubility of D-luciferin potassium salt? Is its solution stable?

The solubility of D-luciferin potassium salt is above 40 mg/ml. Its aqueous solution can be stored at -80°C for a short period without significantly affecting assay results. For some assays that are more sensitive to conditions, it is recommended to freshly prepare the reaction solution to control variable factors in the assays. For example, low levels of luciferase or non-optimal reaction conditions require high-quality substrates for the reaction.

What is the recommended dosage of D-luciferin potassium salt?

For in vivo imaging: The injection amount is determined at 10 μL/g body weight. Each mouse receives 150 mg of D-luciferin potassium salt per kg of body weight (for a mouse weighing 10 g, 100 μL should be injected to provide 1.5 mg of D-luciferin potassium salt). For in vitro imaging: A working solution of 150 μg/ml is generally used for in vitro cell imaging. Tips: The injection method, type of animal, and body weight can all affect the emission of the signal. It is necessary to explore these factors appropriately. Once the fluorescent signal reaches the strongest and most stable plateau phase, imaging analysis should be conducted using the corresponding instruments.

FAQ - Shanghai VKEY Biotechnologies Co.,Ltd.

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FREQUENTLY ASKED QUESTIONS

Do all reagents and samples need to be equilibrated to room temperature when detecting reporter gene?

Yes, under room temperature conditions (20-22°C), reaction components such as cell lysates and substrate working solutions need to be adjusted to room temperature. This is because the reaction rate of luciferase is temperature-dependent. To ensure assay consistency, all reagents and samples should be equilibrated to room temperature.

Can the Firefly Luciferase detection reagents be prepared as a working solution and then aliquoted for long-term storage?

Yes! D-luciferin can slowly degrade in aqueous solution, so it is not recommended to prepare it in advance for long-term storage. If the reagents can be used within six months, they can be prepared as a working solution and then aliquoted and stored at -40°C or below; the lower the temperature, the better the storage stability.

Can the Renilla Luciferase detection reagents be prepared as a working solution and then aliquoted for long-term storage?

No! The substrate for Renilla luciferase (coelenterazine) has very poor stability in aqueous solution and typically degrades within 24 hours. Therefore, Renilla Luciferase detection reagents must be freshly prepared and used immediately.

Can the reporter gene reagents lyse cultured plant cells? Can they lyse primary cells derived from mammalian tissues?

The lysis buffer cannot lyse plant cells. The reporter gene reagents can lyse certain types of mammalian primary cells. Assay testing is required to determine whether a specific cell type can be lysed.

What is the appropriate ratio between the read values of Firefly luciferase and Renilla luciferase?

It is recommended that the read values of Firefly and Renilla luciferases in the control group be similar, or that the read value of Renilla luciferase is slightly higher than that of Firefly luciferase. In actual assays, due to inconsistencies in the quality of plasmid extraction for Firefly luciferase and Renilla luciferase, as well as differences in the regulatory capabilities of the control elements being tested, we recommend conducting preliminary assays to optimize the co-transfection amounts of Firefly luciferase plasmid and Renilla luciferase plasmid.