For Nlux activity screening, 100 μl of cell culture was transferred to a luminescence plate, and 100 μl of F/2 containing Nanoglo substrate (Promega) at a 10,000X dilution. For normalized luminescence measurements, 1.5 million cells per well were transferred to the 96-well luminometer plate, the volume was adjusted to 100 μl with F/2 medium, and 100 μl of F/2 containing Nanoglo substrate (Promega) at a 10,000X dilution was added. Luminescence was measured after 180 sec delay with a 0.3 sec exposure using a Centro XS3 LB960 (Berthold).
Nano glo substrate
Manufactured by Promega
Sourced in United States, Germany, Finland
The Nano-Glo substrate is a luciferase-based reagent used for bioluminescent detection. It provides a robust luminescent signal that can be used to quantify target analytes in a variety of assay formats.
Automatically generated - may contain errors
Lab products found in correlation
Nanoglo lysis buffer, by Promega (4 mentions)
Synergy h4 hybrid microplate reader, by Agilent Technologies (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Living image software, by PerkinElmer (3 mentions)
Orion 2 microplate luminometer, by Berthold Technologies (3 mentions)
Nanoluc, by Promega (3 mentions)
Spark plate reader, by Tecan (3 mentions)
Optiplate, by PerkinElmer (3 mentions)
Casein, by Thermo Fisher Scientific (2 mentions)
Sense beta luminometer, by Promega (2 mentions)
Ctg reagent, by Promega (2 mentions)
Opera qehs, by PerkinElmer (2 mentions)
Mini protean tgx 4 20 gradient gel, by Bio-Rad (2 mentions)
Nanoglo buffer, by Promega (2 mentions)
Halotag nanobret 618 ligand, by Promega (2 mentions)
Factor mix, by New England Biolabs (2 mentions)
Extracellular nanoluc inhibitor, by Promega (2 mentions)
76 protocols using nano glo substrate
1
Nlux Activity Screening in Microalgae
2
Extracellular Vesicle Luciferase Tracking
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To measure the luciferase activity of EVs released by HEK293T cells post-transfection, EVs containing Cas9-Nluc-CRY2 protein were purified from the culture supernatants and resuspended in PBS. The purified EVs were mixed with Nano-Glo substrate diluted in buffer (Promega) and then subjected to luciferase activity detection using EnVision Multimode Plate Reader (PerkinElmer).
To detect the luciferase activity of EVs uptake by recipient cells, isolated EVs were added to Huh7 cells that had been seeded the day before and cultured for 6 h at a 37°C incubator with 5% CO2. After incubation, Huh7 cells were washed with PBS and lysed in 0.1% TritonX-100 in PBS, and then incubated on a shaker for 10 min at room temperature. The luciferase activity of the cell lysates was measured as detailed above.
To determine the distribution of EVs containing Cas9-Nluc-CRY2 in mice after tail vein injection, tissues were harvested and lysed in 0.1% TritonX-100 using the Qiagen Tissue Lyser II according to the manufacturer’s instructions. The tissue lysates were diluted to a suitable concentration and mixed with buffer-diluted Nano-Glo substrate (Promega). The luciferase activity of the tissue lysates was measured as detailed above [24 (link)].
To detect the luciferase activity of EVs uptake by recipient cells, isolated EVs were added to Huh7 cells that had been seeded the day before and cultured for 6 h at a 37°C incubator with 5% CO2. After incubation, Huh7 cells were washed with PBS and lysed in 0.1% TritonX-100 in PBS, and then incubated on a shaker for 10 min at room temperature. The luciferase activity of the cell lysates was measured as detailed above.
To determine the distribution of EVs containing Cas9-Nluc-CRY2 in mice after tail vein injection, tissues were harvested and lysed in 0.1% TritonX-100 using the Qiagen Tissue Lyser II according to the manufacturer’s instructions. The tissue lysates were diluted to a suitable concentration and mixed with buffer-diluted Nano-Glo substrate (Promega). The luciferase activity of the tissue lysates was measured as detailed above [24 (link)].
3
Intranasal Ferret Infection Imaging
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Ferrets infected intranasally with A/California/04/2009-PA NLuc virus were imaged as previously described (45 (link)). Briefly, ferrets were anesthetized with 4% inhaled isoflurane, and the chest area directly above the lung was shaved to minimize background. Ferrets were injected via the cephalic vein with Nano-Glo substrate (Promega N1110) at a dilution of 1:5 in sterile PBS. Ferrets were imaged using a Xenogen IVIS200 (PerkinElmer) with an exposure time of 5 min. Data were analyzed with LivingImage software (PerkinElmer).
4
Monitoring Hsf1 Activity via yNlucPEST
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A yNlucPEST reporter was used to monitor Hsf1 activity as previously described49 (link). Briefly, Nano-Glo substrate (Promega, Madison, WI, USA) was diluted 1:100 with the supplied lysis buffer and mixed 1:10 with logarithmic growing cells carrying the reporter plasmid pCA955. Bioluminescence was determined after 3 min of incubation, using an Orion II Microplate Luminometer (Berthold Technologies GmbH, Bad Wildbad, Germany) and the obtained signal was normalized to OD600.
5
Neutralization Assay for JUNV Pseudotypes
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JUNV trVLPs were generated as previously described [20 (link),21 (link)]. Briefly, BSR-T7/5 cells were transfected with plasmids encoding JUNV NP, JUNV L, a nanoluciferase-expressing JUNV S-segment minigenome, and T7. After 24 h, cells were further transfected with plasmids encoding JUNV Z and either the homologous JUNV (strain Romero) GP or plasmids encoding GPs from different JUNV strains, as indicated (i.e., strain Espindola, P3551, or XJ13). To assess neutralization, trVLP preparations were incubated 1:1 with a two-fold dilution series of heat-inactivated (56 °C, 30 min) serum samples from either human Candid#1 vaccinees or immunized rabbits, as indicated. Samples were incubated for 2 h at 37 °C before 100 µL of the trVLP/serum mixture were applied to Huh7 cells pre-transfected with JUNV NP and JUNV L, as previously described [20 (link)]. For each sample, 4 biological replicates per experiment were performed. Reporter activity was measured after 48 h using NanoGlo substrate (Promega, Madison, WI, USA) on a GloMax Discover microplate reader (Promega, WI, USA).
6
Nano-Glo Bioluminescence Assay Protocol
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Nano‐Glo substrate (Promega GmbH, Germany) was diluted 1:100 with the supplied lysis buffer and mixed 1:10 with cells grown in SC in a white 96‐well plate. Bioluminescence was determined immediately, using an Orion II Microplate Luminometer (Berthold Technologies GmbH & Co. KG, Germany). Bioluminescence light units (BLU) are defined as the relative light units (RLU)/s of 1 ml cells at OD600 = 1.0. To monitor turnover, 1 mg/l cycloheximide (Sigma‐Aldrich, St. Louis, MO, USA) or 5 µm lactimidomycin (Merck‐Millipore) was added. For in vitro bioluminescence measurements, cells were subjected to glass bead lysis for 30 s in a bead beater and cell debris was removed by 10 min centrifugation at 1500 × g. Cell‐free lysates were diluted to a protein concentration of 0.05 mg/ml (Nanodrop, 280 nm) and mixed with cycloheximide before determining bioluminescence.
7
Pseudovirus Infection Luciferase Assay
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Following pseudovirus infection, cells were washed twice with PBS, which was subsequently aspirated. Lysis buffer (Promega, E1531) was added (50 µl/well) and incubated rotating for 15 min at room temperature. NanoGlo Substrate (Promega, N1130) was diluted 1:50 in assay buffer and 25 µl/well was added and incubated for an additional 15 min. Samples were transferred to a white, opaque-bottom 96-well plate and luminescence was read using a BMG Labtech FLUOstar Omega plate reader.
8
Pneumococcal Transformation Efficiency Assay
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Split luciferase assays were carried out as previously described (49 (link)–51 (link, link)), with modifications. Briefly, pneumococcal cells were grown in a C + Y medium (with 50 µM IPTG where required) at 37 °C until OD550 0.1, and competence was induced by the addition of 100 ng mL−1 CSP. The cells were then incubated for 10 min at 37 °C before the addition of R1501 chromosomal DNA (250 ng µL−1) where noted, followed by a further 5 min incubation at 37 °C. The cells were then washed in fresh C + Y medium and 1% NanoGlo substrate (Promega) was added and luminescence was measured 20 times every 1 min in a plate reader (VarioSkan luminometer, ThermoFisher). Data are represented as mean ± SEM calculated from nine independent repeats, with individual data points plotted.
9
Novel LIPS Bridging Assay for High-Throughput
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To develop a novel LIPS bridging assay format for high throughput requirements, Spike antigen diluted to 2.5ng/µl in 40µl PBS was pipetted into every well of a 96-well high-binding OptiPlate™ (Perkin-Elmer, Waltham, MA, USA) and incubated for 18hrs at 4°C. The plate was washed 4 times with TBST and blocked with 1% Casein in PBS (Thermo Scientific, Waltham, MA, USA). The plate was left to air-dry for 2-3hrs before being stored with a sachet of desiccant in a sealed plastic bag at 4°C and used within three weeks.
The Nluc-RBD antigen was diluted in TBST to 10x106+/-5% LU per 25µl. Sera (1.5µl, 2 replicates) were pipetted into a 96-well plate and incubated with 37.5µl diluted labelled antigen for 2hrs. Of this mixture, 26µl was transferred into the coated OptiPlate and incubated shaking (~700rpm) for 1.5hrs. The plate was washed 8 times with TBST, excess buffer was removed by aspiration, then 40µl of a 1:1 dilution of Nano-Glo® substrate (Promega) and 20mM Tris 150mM NaCl pH 7.4 with 0.15% v/v Tween-20 was injected into each well before counting in a Hidex Sense Beta Luminometer (Turku, Finland). Units were interpolated from LU through a standard curve.
The Nluc-RBD antigen was diluted in TBST to 10x106+/-5% LU per 25µl. Sera (1.5µl, 2 replicates) were pipetted into a 96-well plate and incubated with 37.5µl diluted labelled antigen for 2hrs. Of this mixture, 26µl was transferred into the coated OptiPlate and incubated shaking (~700rpm) for 1.5hrs. The plate was washed 8 times with TBST, excess buffer was removed by aspiration, then 40µl of a 1:1 dilution of Nano-Glo® substrate (Promega) and 20mM Tris 150mM NaCl pH 7.4 with 0.15% v/v Tween-20 was injected into each well before counting in a Hidex Sense Beta Luminometer (Turku, Finland). Units were interpolated from LU through a standard curve.
10
Bioluminescence Imaging of Infected Mice
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To perform the bioluminescence imaging, the infected mice were shaved in advance and anaesthetised via the subcutaneous (s.c.) injection of Avertin (150 μl/10 g of 2.5% solution). The Nano-Glo substrate (Promega) was diluted 1:20 in PBS, and each mouse was i.p. injected with 100 μl of the mixture. The bioluminescence data were collected using an IVIS CCD camera system (Caliper Life Science), and further processed in Living Image (version 4.5) software (Caliper Life Sciences). To analyse the bioluminescence signals, the ROIs were selected manually in the uniformly scaled images, and the data were defined as total flux in photons/second.
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