Luciferase Assay Kits: Follow The Light

A short lifetime is perfectly fine if you simply want to take a quick reading with great sensitivity. Perkin Elmer, for example, tries to remedy this problem by incorporating proprietary mixtures into their Britelite plus assay system. Although the proprietary mixture modifies the reaction to lengthen the signal, the Britelite plus system is still better suited to experiments requiring higher sensitivity and a shorter signal half-life. According to Amy McCann, global product leader for luminescence reagents at Perkin Elmer, the Britelite plus system provides “ultra-high sensitivity down to femtomole range, with a signal half-life of 30 minutes.”

Cell viability and cytotoxicity studies also benefit from luciferase tools that favor greater sensitivity over signal half-life. Perkin Elmer’s ATPlite 1step is a luciferase-based ATP monitoring system that can replace other methods used for quantifying the proliferation and cytotoxicity of cultured mammalian cells, such as colorimetric, fluorometric, and radioisotopic assays. The convenience of having only one reagent addition step makes it easy to use, both manually and in robotic systems. The assay yields a linear correlation between cell number and luminescence: up to 50,000 cells per well for 96-well plates, and up to 12,500 cells per well for 384-well plates. McCann says that the ATPlite 1step is “sensitive down to 5 cells in 100 microliters of medium, with a wide linear dynamic range of greater than 5 logs, within a measurement window of zero to 30 minutes.”

In comparison, Neal Cosby, strategic marketing manager for drug screening at Promega, cites their “CellTiter-Glo Cell Viability Assay, [which] exhibits a dynamic range of 8 logs and a sensitivity to as few as 10 cells. Comparable fluorescent assays only show a linear range of one to two logs and sensitivity of about 1000 cells under similar conditions.” Promega’s Glo family of bioluminescent assays is based on a genetically engineered form of fire fly enzyme called “Ultra-Glo Luciferase” that was created by Promega scientists. “Ultra-Glo outperforms other luciferase enzymes and remains active under a wide range of assay conditions, such as variable temperature, varied ionic strength, and prolonged exposure in solution, that diminish the effectiveness of most other assay formats,” says Cosby.

Similar to the Steadylite assay kit, the Luc-Screen Firefly Luciferase Reporter Gene Assay System with Extended-Glow Light Emission Kinetics from Applied Biosystems is engineered to have a particularly long signal half-life for measurement of luciferase activity in culture media of multiwell plates. Suitable for high-throughput applications, this assay takes about one hour to complete. Light emission can be measured within a time window of between about 10 minutes to several hours after reagents are added.

BioAssay Systems’ SuperLight Luciferase Reporter Gene Assay is based on luciferase expression in mammalian, yeast, or E. coli cells, and only requires the addition of a single reagent to the cells, at which point the cells are simultaneously lysed, and then the light intensity measured after a two-minute incubation. All kit components are compatible with culture media and with all liquid handling systems, making the system amenable to scaling up. With an extended signal half-life of 40 minutes, the SuperLight luciferase assays are especially suitable for high-throughput screening in 96-, 384-, and 1536-well plates.

Another system that may help you attain the best of both worlds is Clontech’s Ready-To-Glow Secreted Luciferase System, a reporter system based on a secreted form of luciferase originally cloned from the marine copepod Metridia longa. The gene for this 24 kDa enzyme was optimized by deleting potential splice sites, and increasing the overall GC content to prolong mRNA half-life. This assay is particularly advantageous if you don’t want to lyse your cells—you can monitor promoter activation in live cells by measuring the enzymatic activity in the supernatant. The system is especially useful for studies of signal transduction pathways, where interactions of transcription factors with different promoters and response elements must be monitored. Clontech claims that compared to firefly luciferase, Metridia-secreted luciferase shows a higher signal stability without compromising signal intensity. The Ready-To-Glow Secreted Luciferase Reporter System, which consists of the Ready-To-Glow Secreted Luciferase Reporter Assay and the Ready-To-Glow Secreted Luciferase pMetLuc Vector Kit, is also amenable to high-throughput scaling.

New England BioLabs offers an assay for a secretable form of luciferase, as well, from the copepod Gaussia princes. Their Gaussia Luciferase Assay Kit is designed to measure the enzyme activity in the supernatant of cells in culture without cell lysis. New England BioLabs claims that Gaussia luciferase has a signal intensity that is 1000-fold higher than luciferases from fire fly or sea pansy (Renilla reniformis).

There are more choices than ever before when it comes to the ways in which to perform your high-throughput screening. “The extreme sensitivity and excellent signal-to-noise ratio of bioluminescence assays make them much more suited to miniaturization in uHTS applications,” says Cosby. “Also, most bioluminescent assays can easily be multiplexed with other fluorescent endpoint assays.”