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Neolite luciferase substrate

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The Neolite luciferase substrate is a laboratory product designed to measure luciferase activity. It provides the necessary components for the bioluminescent reaction catalyzed by luciferase enzymes, enabling the quantification of luciferase-based reporter gene expression or ATP levels.

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Lab products found in correlation

Glo lysis buffer, by Promega (4 mentions) Envision plate reader, by PerkinElmer (3 mentions) Victor3 microplate reader, by PerkinElmer (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions) Janus automated liquid handling system, by PerkinElmer (1 mentions) Pin tool, by V&P Scientific (1 mentions) Azidothymidine azt, by Merck Group (1 mentions) Culturplate 384, by PerkinElmer (1 mentions) Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions) Pgl4.32 luc2p nf κb re hygro plasmid, by Promega (1 mentions)

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Luciferase substrate (14 citations) NeoLite (4 citations) Neolite substrate (2 citations)

7 protocols using neolite luciferase substrate

1

Quantification of Neutralizing Antibodies

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Neutralizing antibodies were quantified as reduced luciferase expression levels following H5pp transduction in MDCK cells. 50 μl H5pp (50TCID50) was incubated with 50 μl of antisera (two-fold serial dilution, starting dilution 1∶40) for 1 h at 37°C followed by the addition of MDCK cells (1.5×104 cells/well). At 2 d post-infection, cells were lysed with Glo Lysis Buffer (Promega). Luciferase activity was measured by the addition of neolite luciferase substrate (PerkinElmer). Neutralization titers (IC50) were measured as the serum dilution required to obtain a 50% reduction in RLU compared to control wells containing the virus only.
Lin S.C., Liu W.C., Jan J.T, & Wu S.C. (2014). Glycan Masking of Hemagglutinin for Adenovirus Vector and Recombinant Protein Immunizations Elicits Broadly Neutralizing Antibodies against H5N1 Avian Influenza Viruses. PLoS ONE, 9(3), e92822.
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2

Mycobacterial Luciferase Screening System

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To development a high-quality high-throughput screening (HTS) system, we have inserted luciferase encoding luc gene into an E.coli-Mycobacteria shuttle plasmid pMV261 and constructed recombinant plasmid pMV261-luc. Sequences of primers used to amply luc gene as following: PL1: 5′-CTGCAG(Pst I)ATGGAAGACGCCAAAAAC-3′; PL2: 5′-GTCGAC(Sal I)CAATTTGGACTTTCCGCCC-3′. The recombinant plasmids were transferred to M. Smegmatis mc(2)155 strain when they were isolated from E.coli DH5α. Parameters of electroporation were 1.8kv, 25uF, 200 Ω29 (link). And the positive M. Smegmatis-luciferase clones (named MSL) were incubated with neolite luciferase substrate (PerkinElmer, USA) at room temperature for 60 minutes. Luciferase activity (relative luciferase units, RLU) was measured by an EnVision plate reader (PerkinElmer, USA)30 (link).
Shen H., Wang F., Zeng G., Shen L., Cheng H., Huang D., Wang R., Rong L, & Chen Z.W. (2016). Bis-biguanide dihydrochloride inhibits intracellular replication of M. tuberculosis and controls infection in mice. Scientific Reports, 6, 32725.
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3

NF-κB Activation by Flagellin Proteins

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HEK 293 A cells were co-transfected with 7.5 μg pDUO-hTLR5 plasmid (InvivoGen, San Diego, CA, USA) and 3 μg pGL4.32 [luc2p/NF-ĸB-RE/Hygro] plasmid (Promega, Madison, WI, USA). The transfected cells were seeded at a density of 5 × 104 cells/well in a 96-well plate. The next day, FliC, FliC-ZDIII, FliCΔD3-2ZDIII, and FliCΔD2ΔD3-3ZDIII proteins were serially diluted and incubated with the transfected cells for 5 h at 37 °C. The cells were lysed with Glo-lysis buffer (Promega), and a neolite luciferase substrate (PerkinElmer, Waltham, MA, USA) was added. Luciferase activity was determined using a Victor III microplate reader (PerkinElmer).
Chen C.H., Chen C.C., Wang W.B., Lionel V., Liu C.C., Huang L.M, & Wu S.C. (2022). Intranasal Immunization with Zika Virus Envelope Domain III-Flagellin Fusion Protein Elicits Systemic and Mucosal Immune Responses and Protection against Subcutaneous and Intravaginal Virus Challenges. Pharmaceutics, 14(5), 1014.
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4

siRNA Screening for Virus Inhibition

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All siRNAs were arrayed in 384-well plates. For each assay plate well, 2 μl siRNA was mixed with 0.1 μl Lipofectamine RNAiMAX (Invitrogen) and 9.9 μl Opti-MEM (Invitrogen). After 20 min incubation at room temperature, 38 μl cell suspension of 1000 A549 cells was added, resulting in a final siRNA concentration of 10nM. Cells were incubated at 37 °C and 5 % CO2 for 48 h. Then the medium was removed and the cells were challenged with 30 μl MARV or AIV virions in parallel. Virions were removed 24 h later and 30 μl fresh DMEM supplemented with FBS and antibiotics was added to each well. After another 24 h incubation, 15 μl of neolite luciferase substrate (PerkinElmer, Waltham, MA) was mixed in, incubated for 5 min, and luciferase activity was measured with an Envision plate reader (PerkinElmer). All multi-well pipetting procedures were conducted by the JANUS automated liquid handling system (PerkinElmer).
Cheng H., Koning K., O’Hearn A., Wang M., Rumschlag-Booms E., Varhegyi E, & Rong L. (2015). A parallel genome-wide RNAi screening strategy to identify host proteins important for entry of Marburg virus and H5N1 influenza virus. Virology Journal, 12, 194.
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5

High-throughput Screening for SARS-CoV-2 Inhibitors

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The Chembridge small molecule library (19,200 compounds) was screened following a previously published protocol with modifications. Briefly, the screen was performed using 384-well plates with 320 compounds arrayed in each plate. The following controls were used in the screen: azidothymidine (AZT; Sigma, St. Louis, MO) was used as a positive control and DMSO was used as a negative control.
A549 cells were seeded into white, flat-bottom, 384-well plates (CulturPlate-384; PerkinElmer, Waltham, MA) at a density of ~1000 cells/well in 30-μL assay medium using JANUS liquid handler MDT (Modular Dispense Technology; PerkinElmer) and incubated at 37 °C, 5% CO2. Twenty-four hours later, 0.2 μL of each compound was transferred into wells containing 79.8 μL DMEM in an intermediate 384-well plate through a pin tool (V&P Scientific, San Diego, CA) and mixed thoroughly using a JANUS liquid handler MDT. Twenty μL compound plus 20 μL pseudovirus was transferred to the target cell plates. Plates were incubated at 37 °C, 5% CO2 for 48 h. After incubation, 20 μL of neolite luciferase substrate (PerkinElmer) was added to each well, and plates were incubated at room temperature for 5 to 10 min. Luciferase activity was measured by an EnVision plate reader (PerkinElmer). The data were analyzed using the statistical programming language R as previously described.
Hussein A.F., Cheng H., Tundup S., Antanasijevic A., Varhegyi E., Perez J., AbdulRahman E.M., Elenany M.G., Helal S., Caffrey M., Peet N., Manicassamy B, & Rong L. (2020). Identification of entry inhibitors with 4-aminopiperidine scaffold targeting group 1 influenza A virus. Antiviral research, 177, 104782.
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6

Generating H5 Pseudotyped Particles

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For generating H5pp particles, three plasmids including (i) pcDNA3.1(+) plasmids expressing H5HA of A/Thailand/1(KAN-1)/2004(H5N1) (clade 1), A/bar-headed goose/Qinghai/1A/2005(H5N1) (clade 2.2), A/Hubei/1/2010 (H5N1) (clade 2.3.2.1a), or A/Anhui/1/2005(H5N1) (clade 2.3.4), (ii) pcDNA3.1(+) plasmid expressing N1NA of A/Thailand/1(KAN-1)/2004(H5N1), and (iii) pNL Luc E- R- plasmid were co-transfected to 293T cells as described previously (16 (link), 20 (link)). 50 μl of two-fold serially diluted serum samples (starting at 1:10) were incubated with 1 TCID50 of H5pp at 37°C for 1 h, and then the mixtures were incubated with 1.5×105 MDCK cells per well at 37°C for 48 h. The H5pp-infected cells were lysed using Glo lysis buffer (Promega), and treated with neolite luciferase substrate (PerkinElmer) for luciferase activity measurements. The relative light units (RLU) values developed from the group without serum and the group without H5pp were defined as 0% and 100% neutralization respectively. The RLU values of the other groups were normalized for neutralization percentage calculation.
Chen T.H., Yang Y.L., Jan J.T., Chen C.C, & Wu S.C. (2021). Site-Specific Glycan-Masking/Unmasking Hemagglutinin Antigen Design to Elicit Broadly Neutralizing and Stem-Binding Antibodies Against Highly Pathogenic Avian Influenza H5N1 Virus Infections. Frontiers in Immunology, 12, 692700.
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7

Quantifying TLR5-Mediated NF-κB Activation

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293A cells were cultured overnight in a 10-cm culture dish and then co-transfected with 7.5 μg of pUNO1-hTLR5 plasmid (InvivoGen) and 5.5 μg of pGL4.32 [luc2p/NF-κB-RE/Hygro] plasmid (Promega) using TurboFect transfection reagent (Fermentas). The next day, the transfected cells were seeded at a density of 5 × 104 cells/well in a 96-well plate, followed by the addition of a 10-fold serial dilution of the recombinant FliC protein or the recombinant FliC-DIII fusion proteins, which were first diluted to 1 μg/ml in DMEM. After incubating for 5 h, the cells were disrupted using Glo lysis buffer (Promega) and treated with Neolite luciferase substrate (PerkinElmer). After 5 min, a VICTOR3 Multi-labeled Microplate Reader (PerkinElmer) was used to read the 96-well plate for the measurement of the relative luminescence units (RLUs).
Lin H.H., Yang S.P., Tsai M.J., Lin G.C., Wu H.C, & Wu S.C. (2019). Dengue and Zika Virus Domain III-Flagellin Fusion and Glycan-Masking E Antigen for Prime-Boost Immunization. Theranostics, 9(16), 4811-4826.
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