When looking to perform in vitro cell-based target validation, pathway analysis, and compound screening there are several types of assays to choose from. One question you might ask when evaluating different cell-based reporter assay technologies is, what is better for my research: an assay system that uses fluorescence or an assay system that utilizes bioluminescence? Both technologies can provide researchers with valuable data so let us weigh their advantages and disadvantages.
Fluorescence detection technologies provide great performance and flexibility. Fluorescence happens when a compound called a fluorophore absorbs light energy becoming excited and when the compound returns to its ground state emits light of a longer wavelength. This fluorescent emission can be used as a reporter system to detect various cellular functions. Since a detection reagent is not required this makes fluorescence-base assays highly stable allowing plates to not only be processed in batch, but also the ability for a plate to be read multiple times. Different fluorophores also produce different wavelengths of light allowing researchers using fluorescence assays the ability to multiplex and measure multiple targets on the same plate. This can be done through staggering excitation signals, or simultaneously based on the differential wavelength characteristics. Though when multiplexing fluorescence-based assays, care must be taken to ensure that the multiple reporting emission wavelengths don’t overlap.
Though fluorescence-based assays provide a stable and strong signal the largest disadvantage of this detection method in a reporter assay is background fluorescence. Fluorescence-based reporter assays have to content with naturally occurring fluorescence in mammalian cells which creates a high signal-to-noise ratio. It is also not uncommon for test compounds, whether they are synthetic small molecules or natural products, to exhibit some level of natural fluorescence. This can become a problem for primary screening campaigns or high throughput screening when test compounds are assayed at concentrations 1,000 to 10,000-fold higher than the concentration of natural ligands. Any test compounds producing even low-level fluorescence may have inordinately high background, resulting in false-positives or false-negatives in a functional assay.
Bioluminescence Cell-Based Assays
Bioluminescence technologies such as firefly luciferase reporter assays provide researchers with increased sensitivity and precision compared to fluorescence assays. Bioluminescence is a chemical process in living organisms in which an enzyme breaks down a substrate to produce light. Unlike with fluorescence, there is no inherent background luminescence in mammalian cells, which allows a luciferase assay to be extremely sensitive with a greater dynamic linear-response range. Bioluminescence assays also very rarely encounter test compounds with inherent light emission properties. So besides not having to deal with a high signal-to-noise ratio, luciferase assays do not have to contend with false signals coming from the test compounds themselves. Without false signals coming from the cells or the compounds, not only does a luciferase reporter assay have more sensitivity and dynamic linear-response range, the low signal-to noise ratio also makes it easier for a researcher to properly process the data and render it meaningful.
Though bioluminescent assays are sensitive, precise, and produce clear results, the reactions require a detection reagent and are quick. This can cause problems when trying to run plates in batch. To compensate for this, a luminometer equipped with injectors is necessary to run plates or take time and materials to optimize the assay for use with a specially formulated luciferase detection reagent designed to provide stable light emissions. With either of these additions to a firefly luciferase reporter assay protocol, bioluminescence easily becomes the preferred choice for functional assays.
Firefly Luciferase Reporter Assay Reaction
Bioluminescence has its advantage over fluorescence for those looking for sensitivity, precision, and clear results, but how does the luciferase reaction in the reporter assay work? In firefly luciferase reporter assays, the luciferase catalyzes the mono-oxidation of D-luciferin in a Mg+2-dependent reaction that consumes oxygen and ATP as co-substrates and produces photon emissions (light). So, following ligand-activation, the receptor complex acts to cause expression of the luciferase reporter. Upon addition of a detection reagent, the intensity of light emission from the luciferase reaction can then be quantified using a luminometer. This change in receptor activity directly correlates to the activation status of the specific receptor.
INDIGO’s Firefly Luciferase Reporter Cells
INDIGO Biosciences is a provider of receptor specific reporter assays which utilize bioluminescent firefly luciferase as the detection method. INDIGO’s luciferase reporter cells incorporate the cDNA encoding beetle luciferase, a 62 kD protein originating from the North American firefly (Photinus pyralis). The INDIGO reporter cell systems are engineered to provide optimal assay sensitivity and dynamic range when quantifying receptor activity. INDIGO’s reporter assay system incorporates a detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set. If you are looking to perform cell-based target validation, pathway analysis, or compound screening, INDIGO’s portfolio of all-inclusive firefly luciferase reporter assay kits and services can help.