Luciferase activating reagent

Manufactured by Promega

Luciferase activating reagent is a chemical solution used in bioluminescence-based assays. It is designed to activate luciferase enzymes, which emit light when combined with their substrate. This reagent is a crucial component in various life science applications that rely on luciferase-based detection and quantification.

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Lab products found in correlation

Passive lysis buffer (plb), by Promega (3 mentions) Passive cell lysis buffer, by Promega (2 mentions) Vero cell, by American Type Culture Collection (2 mentions)

Close spelling variants of this product

Luciferase reagent (50 citations)

5 protocols using luciferase activating reagent

Virus Neutralization Assay Protocol

Cited 1 time

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Neutralization assays were performed as previously described (Howell et al., 2016 (link)). Vero cells were seeded at 60,000 cells/well and cultured overnight in EMEM supplemented with 10% fetal bovine serum and 100 I.U./mL penicillin and 100 μg/mL streptomycin at 37°C and 5% CO₂. The next day, a serial dilution of antibody was incubated with pseudotyped virus in serum free EMEM for 1 hour at room temperature before infecting Vero cells at an MOI of 0.04 at 37°C, 5% CO₂ for 1 hour. After infection, 50% v/v EMEM medium supplemented with 2% FBS, and 100 I.U./mL penicillin and 100 μg/mL streptomycin was added to cells. The next day, cells were lysed with Passive Lysis Buffer (Promega) for 30 minutes at room temperature before the addition of the Luciferase Activating Reagent (Promega). The luminesce was read immediately on a Biotek Plate reader. Percent neutralization was calculated based on wells containing virus only. Data was fit to a 4PL curve in GraphPad Prism 6.

Zhao X., Howell K.A., He S., Brannan J.M., Wec A.Z., Davidson E., Turner H.L., Chiang C.I., Lei L., Fels J.M., Vu H., Shulenin S., Turonis A.N., Kuehne A.I., Liu G., Ta M., Wang Y., Sundling C., Xiao Y., Spence J.S., Doranz B.J., Holtsberg F.W., Ward A.B., Chandran K., Dye J.M., Qiu X., Li Y, & Aman M.J. (2017). Immunization-elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability. Cell, 169(5), 891-904.e15.

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Antibody Neutralization Assay for Ebola

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Three-fold dilutions of antibodies (3 μg/ml – 0.0048
μg/ml) were assayed in technical duplicates and incubated with
1×10⁴ RLU/well of Ebola GP-pseudotyped vesicular
stomatitis virus expressing luciferase (IBT Bioservices) for 1 hour at room
temperature before addition to VeroE6 monolayers (6×10⁵cells/well) and incubation at 37 °C for 14–16h. Cells were
lysed using Passive Lysis Buffer (Promega) and luciferase activity was
determined using a luciferase activating reagent (Promega). IC₅₀titers were determined using Prism 8.0.

Gunn B.M., Lu R., Slein M.D., Ilinykh P.A., Huang K., Atyeo C., Schendel S.L., Kim J., Cain C., Roy V., Suscovich T.J., Takada A., Halfmann P.J., Kawaoka Y., Pauthner M.G., Momo M., Goba A., Kanneh L., Andersen K.G., Schieffelin J.S., Grant D., Garry R.F., Saphire E.O., Bukreyev A, & Alter G. (2021). A Fc-Engineering approach to define functional humoral correlates of immunity against Ebola virus. Immunity, 54(4), 815-828.e5.

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Neutralization Assay with Pseudotyped VSV

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Neutralization assays with replication-incompetent pseudotyped vesicular stomatitis virus (VSV) expressing a luciferase reporter and either WT-EBOV GP (GP_WT) or GP containing the E545D mutation (GP_E545D) were performed as described previously (31 (link)). Briefly, a serial dilution of antibody was incubated with pseudotyped virus in serum-free Eagle’s minimum essential medium (EMEM) for 1 h at room temperature before Vero cells were infected at a multiplicity of infection (MOI) of 0.04. One hour following infection, cells were supplemented with 50% (vol/vol) EMEM containing 2% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Twenty-four hours later, cells were lysed for 30 min at room temperature with Passive Lysis Buffer (Promega) before the addition of Luciferase Activating Reagent (Promega). Luciferase luminescence was read using a Biotek Plate Reader, and percent neutralization was calculated based on wells containing virus only.

Banadyga L., Zhu W., Kailasan S., Howell K.A., Franaszek K., He S., Siragam V., Cheng K., Yan F., Moffat E., Cao W., Leung A., Embury-Hyatt C., Aman M.J, & Qiu X. (2021). Atypical Ebola Virus Disease in a Nonhuman Primate following Monoclonal Antibody Treatment Is Associated with Glycoprotein Mutations within the Fusion Loop. mBio, 12(1), e01438-20.

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Filovirus Neutralization Assay in Vero Cells

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Vero cells (ATCC) were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS and P/S at 37 °C and 5% CO₂. Cells were seeded at 60,000 cells/well in a 96-well black, flat-bottom tissue culture plates. The following day, antibodies were diluted in serum-free EMEM supplemented with P/S and mixed with vesicular stomatitis virus lacking G protein and expressing the appropriate filovirus GP (VSV-GP) (for EBOV, SUDV, or EBOV GP-AAA) for 1hr at room temperature. After 1 hr, 100 μL of the virus and antibody was added to the plated Vero cells at an MOI of 0.04. The plates were incubated for 1 hr at 37 °C before the addition of 100 μL of EMEM supplemented with 2% FBS and P/S and overnight incubation. The next day, media and virus were removed from the plates before adding 30 μL of 1 3 passive cell lysis buffer (Promega). Cells were allowed to lyse for 30 min at room temperate before the addition of 30 μL of luciferase activating reagent (Promega). Lumi-nesce was read immediately on a BioMek plate reader. Data were fit to a 4PL curve using Prism GraphPad software. Percent neutralization was calculated based on wells containing virus without antibodies.

Howell K.A., Brannan J.M., Bryan C., McNeal A., Davidson E., Turner H.L., Vu H., Shulenin S., He S., Kuehne A., Herbert A.S., Qiu X., Doranz B.J., Holtsberg F.W., Ward A.B., Dye J.M, & Aman M.J. (2017). Cooperativity Enables Non-neutralizing Antibodies to Neutralize Ebolavirus. Cell Reports, 19(2), 413-424.

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Filovirus Neutralization Assay in Vero Cells

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Vero cells (ATCC) were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS and P/S at 37°C and 5% CO₂. Cells were seeded at 60,000 cells/well in a 96-well black, flat-bottom tissue culture plates. The following day, antibodies were diluted in serum-free EMEM supplemented with P/S and mixed with vesicular stomatitis virus lacking G protein and expressing the appropriate filovirus GP (VSV-GP) (for EBOV, SUDV, or EBOV GP-AAA) for 1hr at room temperature. After 1 hr, 100 μL of the virus and antibody was added to the plated Vero cells at an MOI of 0.04. The plates were incubated for 1 hr at 37°C before the addition of 100 μL of EMEM supplemented with 2% FBS and P/S and overnight incubation. The next day, media and virus were removed from the plates before adding 30 μL of 1 × passive cell lysis buffer (Promega). Cells were allowed to lyse for 30 min at room temperate before the addition of 30 μL of luciferase activating reagent (Promega). Luminesce was read immediately on a BioMek plate reader. Data were fit to a 4PL curve using Prism GraphPad software. Percent neutralization was calculated based on wells containing virus without antibodies.

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