Nano glo luciferase substrate

Manufactured by Promega

Sourced in United States, Germany

Nano-Glo luciferase substrate is a bioluminescent reagent used to detect and quantify luciferase reporter gene activity. It contains furimazine, a proprietary luciferase substrate that emits light upon oxidation catalyzed by luciferase enzymes.

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Nano-Glo Luciferase Assay Substrate Nano-Glo Luciferase Assay Buffer and Substrate Nano-Glo® Luciferase Nano-Glo substrate Nano luciferase substrate Nano-Glo Luciferase substrate Substrates

38 protocols using nano glo luciferase substrate

Pseudotyped Virus Neutralization Assay

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ACE.293T or ACE2.A549 cells were plated in a 96 well tissue culture dish at 1 X 10⁴ cells/well. Cells were infected with pseudotyped virus normalized for RT activity at MOI=0.2. After 2 days, the culture medium was removed, Nano-Glo luciferase substrate (Promega) was added and plate was read in an Envision 2103 microplate luminometer (PerkinElmer). To determine neutralizing antibody titers, sera or monoclonal antibodies were serially diluted 2-fold and then incubated with pseudotyped virus (approximately 2.5 X 10⁷ cps) for 30 minutes at room temperature and then added to target cells. After 2 days, medium was removed, same amount of fresh medium and Nano-Glo luciferase substrate (Promega) was added and transferred to a microtiter plate which was read in an Envision 2103 microplate luminometer (PerkinElmer).

Tada T., Zhou H., Dcosta B.M., Samanovic M.I., Mulligan M.J, & Landau N.R. (2021). Partial resistance of SARS-CoV-2 Delta variants to vaccine-elicited antibodies and convalescent sera. iScience, 24(11), 103341.

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SARS-CoV-2 Pseudovirus Neutralization Assay

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SARS-CoV-2 pseudovirus harboring Nanoluc luciferase gene was generated by transient transfection of pHIV-1 NL4.3Δenv-Luc and Spike-Δ19-D614G or Spike Δ19-D614G plasmids with VOC mutations plasmids in HEK293T cells using Profection mammalian transfection kit (Promega Inc., Madison, WI, USA) according to the manufacturer’s protocol. After 48 h, pseudoviruses were harvested from the culture supernatant (600× g, 10 min), filtered, and stored at −80 °C until further use. ACE2 and TMPRSS2 receptors expressing HEK293T cells were grown in 10% FBS (Fetal Bovine Serum) supplemented DMEM (Gibco) with penicillin-streptomycin (100 U/mL). Heat inactivated sera were serially diluted and mixed with pseudoviruses for 1 h at 37 °C. Finally, 1 × 10⁴ HEK293T-ACE2-TMPRSS2 cells were added and incubated for 48 h at 37 °C with 5% CO2. Post incubation, nano-Glo luciferase substrate (Promega Inc.) was added (50 µL) and 80 µL lysate was transferred to flat-bottom white plates for luminescence measurement using Cytation-5 multimode reader. Serum dilution at which 50% reduction in infection was observed was determined as the pseudovirus neutralization titer (ID₅₀).

Khaleeq S., Sengupta N., Kumar S., Patel U.R., Rajmani R.S., Reddy P., Pandey S., Singh R., Dutta S., Ringe R.P, & Varadarajan R. (2023). Neutralizing Efficacy of Encapsulin Nanoparticles against SARS-CoV2 Variants of Concern. Viruses, 15(2), 346.

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Bioluminescent Imaging of Bacterial Colonization

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All the animal-related procedures were performed strictly according to the guideline of Tianjin Committee of Use and Care of Laboratory Animals, and experimental protocols were approved by the Animal Ethics Committee of Nankai University. All animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg kg⁻¹) before imaging. Animals received substrates and bacteria by gastric inoculation before imaging, furimazine (1/20 dilution of Nano-Glo® Luciferase substrate, 200 µL gavage volume, Promega) is the substrate for NLuc system and d-luciferin (150 mg kg⁻¹, PerkinElmer) is for FLuc and CBRLuc systems.^{35,36 (link)} Bioluminescence images were acquired at room temperature on a Berthold NightOWL LB 983 Imaging System (Berthold Technologies, Bad Wildbad, Germany). The images were acquired with open filter and exposure time ranges from 1 to 3 min according to the signal intensity, the luminescence counts acquired under different exposure time were all normalized as the average value (total counts/exposure time) to ensure the reasonable comparison, and the pseudo colour images represented the light intensity (from red for most intense to blue for least intense). All bioluminescent mice had a region of interest (ROI), corresponding to the location of bioluminescent strain, which is manually determined.

Zhao N., Liu J.M., Liu S., Ji X.M., Lv H., Hu Y.Z., Wang Z.H., Lv S.W., Li C.Y, & Wang S. (2020). A novel universal nano-luciferase-involved reporter system for long-term probing food-borne probiotics and pathogenic bacteria in mice by in situ bioluminescence imaging. RSC Advances, 10(22), 13029-13036.

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SARS-CoV-2 Spike Protein Pseudotyped Virus Neutralization

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SARS-CoV-2 spike protein-pseudotyped lentiviral stocks were produced by the cotransfection of 293T cells with pMDL, pLenti.GFP-NLuc, pcCoV2.S-Δ19 (or variants thereof), and pRSV.Rev as previously described (41 (link)). Virus stocks were normalized by real-time PCR reverse transcriptase (RT) activity (51 (link)). Pseudotyped virus infections were done with 1 × 10⁴ cells/well in 96-well tissue culture dishes at a multiplicity of infection (MOI) of 0.2 as previously described (41 (link)). Luciferase activity was measured after 2 days using the Nano-Glo luciferase substrate (Promega), and plates were read in an Envision 2103 microplate luminometer (PerkinElmer). To quantify antibody neutralization, sera were serially diluted 2-fold and incubated for 30 min at room temperature with pseudotyped virus (corresponding to approximately 2.5 × 10⁷ cps luciferase) in a volume of 50 μl. The mixture was added to 1 × 10⁴ ACE2.293T cells (corresponding to an MOI of 0.2) in a volume of 50 μl in a 96-well culture dish. After 2 days, the medium was removed, and the Nano-Glo luciferase substrate (Nanolight) was added to wells. Luminescence was read in an Envision 2103 microplate luminometer (PerkinElmer).

Tada T., Dcosta B.M., Samanovic M.I., Herati R.S., Cornelius A., Zhou H., Vaill A., Kazmierski W., Mulligan M.J, & Landau N.R. (2021). Convalescent-Phase Sera and Vaccine-Elicited Antibodies Largely Maintain Neutralizing Titer against Global SARS-CoV-2 Variant Spikes. mBio, 12(3), e00696-21.

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BRET Saturation Assay for RXFP1 and GABAB1

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HEK293T cells were prepared for assay the same as above (HiBiT receptor expression assays), but co‐transfected with HiBiT/Nanoluc‐ and mCitrine‐tagged receptor DNA. For saturation‐style BRET assays 10 ng per well of HiBit/Nanoluc‐labeled RXFP1, or 5 ng per well of HiBit‐labeled GABA_B1, and increasing amounts (0‐4000 ng) mCitrine‐labeled receptor per well were transfected, with empty pcDNA3.1 vector included to make up equal total DNA amounts. BRET measurements were taken immediately after addition of HiBiT complementation reagent. For experiments where Nanoluc was used, PRF‐DMEM containing a 1:250 dilution of Nano‐Glo® luciferase substrate (Promega) was used instead of HiBiT complementation reagent. The BRET ratio was defined as the filtered light emission intensity at 520‐620 nm divided by the emission intensity at 410‐490 nm and measured on a Polarstar Omega platereader (BMG Labtech) at 37°C. BRET unit was defined as the BRET ratio of wells transfected with both donor and acceptor tagged receptor, minus the BRET ratio obtained in cells transfected with Nanoluc/HiBiT‐tagged receptors and the appropriate untagged receptor.

Hoare B.L., Kocan M., Bruell S., Scott D.J, & Bathgate R.A. (2019). Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis. Pharmacology Research & Perspectives, 7(4), e00513.

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Protein-Protein Interaction Assay

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30,000 cells/well were seeded in triplicate in a white opaque flat bottom 96-well plate (Greiner Bio One, Monroe, NC, United States) and allowed to attach overnight. The next morning, the medium was aspirated and 90 μL/well Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA, United States) was added. Each well was transfected with 50 ng FKBP-SmBit and 50 ng FRB-LgBit (Promega, Madison, WI, United States) using 0.3 μL/well Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, United States) transfection reagent according to manufacturer’s protocol. One-day post-transfection, cells were treated with 1 mM amiloride (Sigma-Aldrich, St. Louis, MO, United States) for 30 min. Following the above treatment, 25 μL/well Nano-Glo^® luciferase substrate (Promega, Madison, WI, United States) was added and the plate was equilibrated to room temperature. Cells were treated with either 30 nM Rapa/DMSO or 30 nM FAF/Rapa and immediately placed in a plate reader to measure luminescence with 1 s integration time. Measurements were taken in kinetic mode for 2 h at 30-s intervals.

Peddi S., Pan X, & MacKay J.A. (2018). Intracellular Delivery of Rapamycin From FKBP Elastin-Like Polypeptides Is Consistent With Macropinocytosis. Frontiers in Pharmacology, 9, 1184.

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Neutralization of SARS-CoV-2 using Entry-Inhibition Assay

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Neutralization of authentic SARS-CoV-2 by entry-inhibition assay Neutralization was determined using SARS-CoV-2-Nluc, an infectious clone of SARSCoV-2 (based on strain 2019-nCoV/USA_WA1/2020) which encodes nanoluciferase in place of the viral ORF7 and demonstrated comparable growth kinetics to wildtype virus (Xie et al., 2020 (link)). Vero E6 cells were seeded into black-walled, clear-bottom 96-well plates at 2 × 104 cells/well and cultured overnight at 37°C. The next day, 9-point 4-fold serial dilutions of mAbs were prepared in infection media (DMEM + 10% FBS). SARS-CoV-2-Nluc was diluted in infection media at a final MOI of 0.1 or 0.01 PFU/cell, added to the mAb dilutions and incubated for 30 min at 37°C. Media was removed from the Vero E6 cells, mAb-virus complexes were added and incubated at 37°C for 6 or 24 h. Media was removed from the cells, Nano-Glo luciferase substrate (Promega) was added according to the manufacturer’s recommendations, incubated for 10 min at room temperature and the luciferase signal was quantified on a VICTOR Nivo plate reader (Perkin Elmer).

McCallum M., De Marco A., Lempp F.A., Tortorici M.A., Pinto D., Walls A.C., Beltramello M., Chen A., Liu Z., Zatta F., Zepeda S., di Iulio J., Bowen J.E., Montiel-Ruiz M., Zhou J., Rosen L.E., Bianchi S., Guarino B., Fregni C.S., Abdelnabi R., Foo S.Y., Rothlauf P.W., Bloyet L.M., Benigni F., Cameroni E., Neyts J., Riva A., Snell G., Telenti A., Whelan S.P., Virgin H.W., Corti D., Pizzuto M.S, & Veesler D. (2021). N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2. Cell, 184(9), 2332-2347.e16.

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Luciferase Assay for T Cell Activation

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Raw264.7-H2Kb cells were primed and conditioned the same as above in culture-treated white 96-well plates, media was removed, and cells were washed for at least six times with PBS containing 0.1% BSA. The same number of Jurkat-OT1-NFAT-Luc reporter cells were added and co-cultured with Raw264.7-H2Kb for 20 h. Recombinant murine GMCSF (400 U/mL = 20 ng/mL) was added in the co-culture media. After incubation, the same volume of Nano-Glo luciferase substrate (Promega, US) was added and mixed with cell suspension for luminescence detection.

Lv X., Martin J., Hoover H., Joshi B., Wilkens M., Ullisch D.A., Leibold T., Juchum J.S., Revadkar S., Kalinovska B., Keith J., Truby A., Liu G., Sun E., Haserick J., DeGnore J., Conolly J., Hill A.V., Baldoni J., Kensil C., Levey D., Spencer A.J., Gorr G., Findeis M, & Tanne A. (2024). Chemical and biological characterization of vaccine adjuvant QS-21 produced via plant cell culture. iScience, 27(3), 109006.

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Neutralization of SARS-CoV-2 by Entry-Inhibition

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Neutralization of authentic SARS-CoV-2 by entry-inhibition assay was determined using SARS-CoV-2-Nluc, an infectious clone of SARSCoV-2 (based on strain 2019-nCoV/USA_WA1/2020) which encodes nanoluciferase in place of the viral ORF7 and demonstrated comparable growth kinetics to wildtype virus^{58 (link)}. Vero E6 cells were seeded into black-walled, clear-bottom 96-well plates at 2 × 104 cells/well and cultured overnight at 37 °C. The next day, 9-point 4-fold serial dilutions of mAbs were prepared in infection media (DMEM + 10% FBS). SARS-CoV-2-Nluc was diluted in infection media at a final MOI of 0.01 PFU/cell, added to the mAb dilutions and incubated for 30 minutes at 37 °C. Media was removed from the Vero E6 cells, mAb-virus complexes were added and incubated at 37 °C for 24 hours. Media was removed from the cells, Nano-Glo luciferase substrate (Promega) was added according to the manufacturer’s recommendations, incubated for 10 minutes at room temperature and the luciferase signal was quantified on a VICTOR Nivo plate reader (Perkin Elmer).

Tortorici M.A., Czudnochowski N., Starr T.N., Marzi R., Walls A.C., Zatta F., Bowen J.E., Jaconi S., di iulio J., Wang Z., De Marco A., Zepeda S.K., Pinto D., Liu Z., Beltramello M., Bartha I., Housley M.P., Lempp F.A., Rosen L.E., Dellota E J.r., Kaiser H., Montiel-Ruiz M., Zhou J., Addetia A., Guarino B., Culap K., Sprugasci N., Saliba C., Vetti E., Giacchetto-Sasselli I., Silacci Fregni C., Abdelnabi R., Caroline Foo S.Y., Havenar-Daughton C., Schmid M.A., Benigni F., Cameroni E., Neyts J., Telenti A., Snell G., Virgin H.W., Whelan S.P., Bloom J.D., Corti D., Veesler D, & Pizzuto M.S. (2021). Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody. bioRxiv.

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Serological Profiling of CMV Antibodies

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ELISA kits SmartEIA CMV IgG and EIA CMV IgM (both from TestLine Clinical Diagnostics s.r.o.) were used to measure the IgG and IgM antibodies to CMV with the plasma dilution of 1:101. LIPS profiling of CMV antibodies with pp150 protein fragments has been reported earlier (Burbelo et al., 2009 (link)). Two fragments of immunodominant regions of CMV antigen pp150 were cloned into pNanoLuc vector, and LIPS was performed as reported earlier (Haljasmägi et al., 2020 (link)). The HEK293 cell lysates containing NanoLuc‐fusion proteins (0.5–1 × 10⁶ luminescence units; LU) were incubated with plasma samples and Protein G Sepharose beads (Creative BioMart) to capture antibodies (in 1:40 dilution). After washing, the substrate was added (Nano‐Glo™ Luciferase Substrate, Promega), and luminescence was measured in VICTOR X Reader (PerkinElmer Life Sciences).

Salumets A., Tserel L., Rumm A.P., Türk L., Kingo K., Saks K., Oras A., Uibo R., Tamm R., Peterson H., Kisand K, & Peterson P. (2022). Epigenetic quantification of immunosenescent CD8+ TEMRA cells in human blood. Aging Cell, 21(5), e13607.

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