Supernatants from infected cells were overlaid on top of L929 IFN reporter cells containing the ISRE-luciferase construct (32 (link)) and incubated for 4 h (48- or 96-well plates). The reporter cells were lysed in 5 × Reporter Lysis Buffer (Promega, Madison, WI, USA) for 30 min at room temperature and mixed with firefly luciferin substrate (Promega, Madison, WI, USA), and luminescence was measured on an illuminometer (TECAN).
Firefly luciferin substrate
Manufactured by Promega
Sourced in United States
The Firefly luciferin substrate is a chemical compound used in bioluminescence assays. It serves as a substrate for the enzyme luciferase, which catalyzes the oxidation of luciferin to produce light. This light emission can be detected and quantified to measure the presence or activity of luciferase in various experimental systems.
Automatically generated - may contain errors
6 protocols using firefly luciferin substrate
1
IFN-Responsive Reporter Assay
2
Quantifying Type I Interferon Response
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Cell culture supernatants from transfected cells were overlaid on top of HEK293 IFN reporter cells containing ISRE-luciferase construct (21 (link)) and incubated for 4 h. The reporter cells were lysed in passive lysis buffer (Promega, Madison, WI, USA) for 30 min at room temperature, mixed with firefly luciferin substrate (Promega, Madison, WI, USA), and measured using an illuminometer (Beckman Coulter Inc., Fullerton, CA, USA).
3
In Vivo Bioluminescence Imaging
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We intraperitoneally injected firefly luciferin substrate (0.1 mL at 30 mg/mL, Promega) into recipient mice and waited 5 minutes prior to imaging. The Xenogen IVIS imaging system was used, and data were analyzed using the Living Image 3.0 Software.
4
IFN Reporter Assay for Cell Supernatants
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Cell culture supernatants from S1-treated splenocytes were incubated for 4 h with L929 IFN reporter cells containing the ISRE-luciferase construct. The reporter cells were lysed using passive lysis buffer (Promega, USA), mixed with firefly luciferin substrate (Promega, USA), and measured using an illuminometer (Beckman, USA).
5
IFN-induced luciferase reporter assay
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Cell-culture supernatants from stimulated cells or sera from infected mice were incubated with L929 cells containing a stable IFN-stimulated response element-luciferase reporter plasmid [ISRE-luc (35 (link))] for 4 h. The reporter cells were lysed in Passive Lysis Buffer (Promega, Madison, WI, USA) for 30 min at room temperature, mixed with firefly luciferin substrate (Promega), and measured on a luminometer (Becman coulter, Fullerton, CA, USA).
6
Quantifying Type I IFN Activity in pDCs
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Type I IFN activity in the supernatant of murine pDCs was measured using L929 cells transfected with an interferon-sensitive luciferase construct (ISRE-L929) (46 (link)) with reference to a recombinant mouse IFN-β standard (Sigma). In short, pDC culture supernatants were added to ISRE-L929 IFN reporter cells and incubated for 4–6 h. Then, the cells were lysed in Passive Lysis Buffer (Promega), mixed with firefly luciferin substrate (Promega), and measured on a Victor3 Luminometer.
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