90 ug of enhancer constructs and 10 ug of firefly luciferase plasmid (pGL4.51[luc2/CMV/Neo] (Promega E132A) (normalization control) were co-electroporated into C57/BL6 retinae that were dissected at P0. Retinae were cultured in explant culture media for 6 days. Then retinae were dissociated and Luciferase assay were performed using Nano-Glo Dual-Luciferase Reporter (NanoDLR) assay system (Promega N1610) according to the manufacturer’s protocol. Luminescence measurements were taken as singlets using a SpectraMax M5 (Molecular Devices) plate reader. Experiments were performed in quadruplicates.
Nano glo dual luciferase reporter nanodlr assay system
Manufactured by Promega
Sourced in United States
The Nano-Glo Dual-Luciferase Reporter (NanoDLR) assay system is a luminescent reporter assay that enables the detection and quantification of firefly and Renilla luciferase activities in a single sample. The assay utilizes engineered luciferase enzymes and a proprietary buffer system to provide a sensitive, stable, and rapid luminescent readout.
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4 protocols using nano glo dual luciferase reporter nanodlr assay system
1
Enhancer-Driven Luciferase Expression in Retinae
2
Dual-luciferase Bioluminescence Assay
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Bioluminescence
was measured
using the Nano-Glo Dual-luciferase Reporter (NanoDLR) assay system
(Promega), according to the manufacturer’s instructions. Briefly,
cultures were equilibrated at room temperature. Culture medium was
replaced with 50 μL of PBS/well (in 96-well plates). 50 μL
of OneGlowEx was added and mixed on an orbital shaker (±400 rpm)
for 3–5 min at room temperature. Fluc activity was then measured
by a Victor Multilabel Plate reader (PerkinElmer) at 1 s/well. Subsequently,
50 μL of NanoDLR Stop & Glo Reagent (diluted 1:100) was
added per well, mixed for 10 min on an orbital shaker (±600 rpm)
before measuring Nluc activity by the Victor Multilabel Plate reader
(PerkinElmer) at 1 s/well. For bioluminescence measurements in 384-well
plates reagent amounts were halved. Data were analyzed using Prism
8.0 (GraphPad Software).
was measured
using the Nano-Glo Dual-luciferase Reporter (NanoDLR) assay system
(Promega), according to the manufacturer’s instructions. Briefly,
cultures were equilibrated at room temperature. Culture medium was
replaced with 50 μL of PBS/well (in 96-well plates). 50 μL
of OneGlowEx was added and mixed on an orbital shaker (±400 rpm)
for 3–5 min at room temperature. Fluc activity was then measured
by a Victor Multilabel Plate reader (PerkinElmer) at 1 s/well. Subsequently,
50 μL of NanoDLR Stop & Glo Reagent (diluted 1:100) was
added per well, mixed for 10 min on an orbital shaker (±600 rpm)
before measuring Nluc activity by the Victor Multilabel Plate reader
(PerkinElmer) at 1 s/well. For bioluminescence measurements in 384-well
plates reagent amounts were halved. Data were analyzed using Prism
8.0 (GraphPad Software).
3
Doxycycline-Regulated Luciferase Assay
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3 × 104 HepG2 cells or 4 × 104 freshly isolated rat hepatocytes were seeded in each well of 96‐well plates. Cells were transfected with 100 ng of the constitutive pAAV‐HLP‐Luc or pAAV‐Tetoffbidir‐Alb‐luc and 25 ng of pSV40‐NlucP (ratio 3:1) using Lipofectamine 3000 reagent (Thermo‐Fisher Scientific, Waltham, MA, USA). Various concentrations of doxycycline were added at the indicated times and luciferase assays using the Nano‐Glo® Dual‐Luciferase® Reporter (NanoDLR™) Assay System (Promega, Madison, WI, USA) were performed at the indicated time points, in accordance with the manufacturer's instructions. Cell culture experiments were performed once in triplicate.
4
Enhancer-Driven Luciferase Expression in Retinae
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