Luciferase Cell Line Application
I. Application Principles of Luciferase Cell Lines
Luciferase cell line is stably overexpressing luciferase. In vivo imaging technology allows for non-invasive, direct observation of various biological processes in live animals, including tumor growth, metastasis, disease progression, and gene expression changes. This makes it particularly suitable for quantitative analysis of in vivo tumor growth. In vivo imaging reduces animal costs and enables non-invasive tracking of biological behaviors in small animals, making it widely used in tumor research. Current in vivo bio-imaging technologies include ultrasound, CT, MRI, PET, as well as bioluminescence and fluorescence. Bioluminescence uses the luciferase gene to label cells or DNA, while fluorescence technology uses fluorescent reporter groups such as GFP, RFP, Cy dyes, and others for labeling.Luciferase bioluminescence does not require excitation light but does need the substrate luciferin. In the presence of oxygen and ATP, when the exogenous substrate luciferin is administered (via intraperitoneal or intravenous injection), luciferase catalyzes an oxidation reaction within minutes, producing oxyluciferin and generating light emission. This luminescence only occurs in living cells, and the light intensity correlates linearly with the number of labeled cells. In animals, light is scattered and absorbed during transmission; visible light is primarily absorbed by hemoglobin, mainly in the blue-green spectrum. However, in the red light region above 600 nm, hemoglobin absorption is relatively low. This means that a large portion of the light, despite some scattering, can penetrate tissues and skin and be detected by high-sensitivity CCD cameras. By selecting appropriate CCD lenses and imaging dark boxes, the light intensity can be quantified by the instrument, reflecting the number of cells. This technology offers convenient operation, rapid measurement, clear images, low signal-to-noise ratio, cost-effectiveness, and accurate quantification, making it widely accepted by researchers and extensively applied in animal studies.
II. Common Questions on Luciferase Cell Line Application
1. Does luciferase expression affect cell function?Luciferin does not affect the normal physiological functions of animals.
2. Is luciferase stable?
For mammalian bioluminescence, the luciferase gene is typically integrated into the chromosomal DNA of cells to express luciferase, creating cell lines that stably express luciferase. This expression remains continuous and stable as cells divide, metastasize, or differentiate.
3. Why is antibiotic selection frequently used during in vitro cell culture?
Luc cells can stably express luciferase. During in vitro culture, selection pressure is not required as long as culture duration is not prolonged and passage numbers are not high. No gene loss has been reported to date. However, to maintain the luminescence intensity, it is recommended to use antibiotic selection periodically.
4. Why is no luminescence observed during in vivo imaging, or is the luminescence very weak?
· Instrumentation and Equipment: Specialized lenses and light-tight dark boxes are needed to block external light. The sensitivity of the instrument for detecting luminescent cells is related to luminescence intensity. Two key factors enable visible light imaging to detect weak in vivo luminescence: highly sensitive cooled CCD cameras (reaching temperatures as low as -90°C) that can detect even very few photons emitted in vivo, and absolutely sealed dark box systems that block all light, including cosmic rays.
· Luciferin Administration: Luciferin diffuses rapidly and can be administered via intraperitoneal or tail vein injection. Intraperitoneal injection has slower diffusion and longer-lasting luminescence. After intraperitoneal injection, cells expressing luciferase begin glowing approximately 1 minute later, reaching a stable peak intensity at about 10 minutes, maintaining that peak for about 20-30 minutes before starting to decay. Luciferin is excreted within about 3 hours, after which luminescence ceases. The optimal detection window is 15-35 minutes post-injection. Intravenous injection offers rapid diffusion but shorter luminescence duration. Based on extensive experimental data, the recommended luciferin dose is 150 mg/kg (i.e., 3 mg for a 20g mouse).
· Cell Detection Limits: In vivo bioluminescence imaging can detect as few as several hundred subcutaneous cells. The minimum detectable cell number varies with the depth of the luminescent source within the mouse. At the same depth, detected luminescence intensity has an excellent linear correlation with cell number, allowing instrument quantification to reflect cell quantity. Under optimal labeling conditions, instruments can detect a minimum of 100 subcutaneously implanted luminescent cells. For cells located deeper (e.g., orthotopic implantation in internal organs), a higher number of cells is required for detection.
· Other Factors: Different brands of luciferin substrates may have different metabolic processes; it is advisable to conduct preliminary in vitro experiments and establish a standard curve. Other influencing factors include tumor formation time and mouse strain. Tumor formation requires time; nude mice and immunodeficient mice are commonly used and more susceptible to tumor formation.
5. Why are in vitro luminescence values low?
· Detection Timing: The in vitro half-life of luciferase is generally about 30 minutes. Detection should be performed immediately after substrate addition, ideally within 30 minutes.
· Detection Plates: To prevent cross-well interference, use opaque white 96-well plates. Black plates can be used but may reduce signal due to light absorption.
· Instrument Settings: Microplate readers typically do not require wavelength selection; choosing the appropriate filter is sufficient.
· Luciferin Substrate: Different substrates require specific storage conditions; keep luciferin protected from light and sealed. Firefly luciferin substrate should be stored at -20°C. Ensure a sufficient, saturating amount of substrate is added, as insufficient substrate can cause significant deviations in results.
6. Why is depilation necessary for mouse imaging?
Mouse fur scatters, absorbs, and blocks light, and also produces strong autofluorescence. If not removed, fur creates a strong background signal during imaging.
7. Can luminescence be detected from deep within animal tissues?
The minimum detectable cell number varies with the depth of the luminescent source. Generally, each centimeter of tissue depth attenuates luminescence intensity by approximately 10-fold. Luciferase labeling is suitable for in vitro detection or subcutaneous detection and is not ideal for labeling cells in deep organs. However, in mice, if luminescent cells are sufficiently bright, tumors can be detected anywhere within the mouse, as light can penetrate up to 3-4 cm. The typical thickness of a mouse is 7-8 cm.
8. Besides tracking tumor cell behavior, can luciferase technology be used in other research areas?
Yes, this technology enables rapid measurement of tumor growth and metastasis in various cancer models, allowing real-time assessment of changes in tumor cells. It can also be applied in stem cell tracking, leukemia research, bacterial labeling, studies of gene expression in apoptosis, protein interactions, tissue-specific gene expression, and investigations into protein-protein interactions.
