title: A cooled CCD camera-based protocol provides an effective solution for in vitro monitoring of luciferase.
abstract: Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes. However, the mainstay protocols require a luminometer to acquire and process the data, therefore limiting its application to specialized research labs. To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD), instead of a luminometer for data acquiring and processing. By measuring activities of different luciferases, we characterized their substrate specificity, assay linearity, signal-to-noise levels, and fold-changes via CCCD. Next, we defined the assay parameters that are critical for appropriate use of CCCD for different luciferases. To demonstrate the usefulness in cultured mammalian cells, we conducted a case study to examine NFκB gene activation in response to inflammatory signals in human embryonic kidney cells (HEK293 cells). We found that data collected by CCCD camera was equivalent to those acquired by luminometer, thus validating the assay protocol. In comparison, The CCCD-based protocol is readily amenable to live-cell and high-throughput applications, offering fast simultaneous data acquisition and visual and quantitative data presentation. In conclusion, the CCCD-based protocol provides a useful alternative for monitoring luciferase reporters. The wide availability of CCCD will enable more researchers to use luciferases to monitor and quantify biological processes.
authors: Department of Biological Sciences, California State University, East Bay, 25800 Carlos Bee Blvd, Hayward, CA 94542, USA. Electronic address: Afshari.amirali@gmail.com.
authors: Amirali
authors: A
authors: Afshari
authors: Department of Biological Sciences, California State University, East Bay, 25800 Carlos Bee Blvd, Hayward, CA 94542, USA. Electronic address: Claudia.stone@csueastbay.edu.
authors: Claudia
authors: C
authors: Uhde-Stone
authors: System Biosciences (SBI), 265 North Whisman Rd., Mountain View, CA 94043, USA. Electronic address: BLu@systembio.com.
authors: Biao
authors: B
authors: Lu
mesh_terms: D000818:Animals; D048888:Fireflies / Q000201:enzymology; D017930:Genes, Reporter; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000295:instrumentation; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis* / Q000235:genetics; D049410:Luciferases, Renilla / Q000032:analysis* / Q000235:genetics; D049408:Luminescent Agents / Q000032:analysis* / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D048790:Renilla / Q000201:enzymology; D014162:Transfection
chemical_list: D049408:Luminescent Agents; D011994:Recombinant Proteins; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly
keywords: Cooled charge coupled device (CCCD); Firefly luciferase; Gaussia luciferase; High-throughput screening; Renilla luciferase