Nano glo luciferase assay reagent

Manufactured by Promega

Sourced in United States

The Nano-Glo® Luciferase Assay Reagent is a bioluminescent detection system that enables the measurement of nanomolar concentrations of NanoLuc® luciferase in cell-based assays. It provides a sensitive and quantitative way to detect and monitor NanoLuc® reporter gene activity.

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Lab products found in correlation

Nanoluc luciferase, by Promega (4 mentions) Ppk cmv f4 fusion vector, by PromoCell (4 mentions) Prism 9, by GraphPad (3 mentions) Clariostar plus microplate reader, by BMG Labtech (3 mentions) Victor x multilabel plate reader, by PerkinElmer (3 mentions) 96 well microfilter plate, by Merck Group (3 mentions) 37 c envision plate reader, by PerkinElmer (3 mentions) Nano glo luciferase assay substrate, by Promega (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) M1000, by Tecan (2 mentions) Microplate reader, by Tecan (2 mentions) Microplate luminometer, by Promega (1 mentions) Vigofect transfection reagent, by Vigorous Biotechnology (1 mentions) Glomax discover system, by Promega (1 mentions) Synergy h1, by Agilent Technologies (1 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Hek293 cell, by American Type Culture Collection (1 mentions) Calfectin, by SignaGen (1 mentions) Spectramax i3 plate reader, by Molecular Devices (1 mentions)

Spelling variants of this product

Luciferase assay reagent (446 citations) Luciferase assay (182 citations) Nano-Glo (59 citations) Luciferase reagent (50 citations) Nano-Glo luciferase reagent (19 citations) Nano-Glo® Luciferase (14 citations)

51 protocols using nano glo luciferase assay reagent

SARS-CoV-2 Virus-Like Particle Production

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Adherent HEK293T cells at 80% confluence were co-transfected with equimolar amounts of plasmids encoding the CoV-S, E, M, and HiBiT-N proteins using LipoD transfection reagent (SignaGen, Frederick, MD, USA). 1:3 DNA:LipoD mixtures were mixed in serum free-DMEM for 10 min at room temperature followed by dropwise addition onto cells. Four h later, media were replenished with DMEM-1% FBS. Media containing VLPs were collected at 24 h post-transfection, clarified by sequential centrifugation, first at 300× g, 4 °C, 10 min, then at 3000× g, 4 °C, 10 min. VLPs in media were then concentrated 20-fold by ultrafiltration using 100 K Amicon ultra filters (MilliporeSigma, St. Louis, MO; USA), and 0.5-mL volumes were then applied to size-exclusion chromatography (SEC) columns. Fractions were collected according to manufacturer’s instructions (Izon Science Ltd., Medford, MA, USA), and VLPs were identified in eluted fractions using Nano-Glo luciferase assay reagents (Promega, Inc.). Briefly, aliqouts of each fraction were mixed with LgBiT protein in the presence of Nano-Glo luciferase assay substrate (Promega), and Nluc enzyme levels were measured in terms of relative light units (RLU) using a Veritas microplate luminometer. Fractions containing HiBiT-VLPs were aliquoted and stored at −80 °C.

Kumar B., Hawkins G.M., Kicmal T., Qing E., Timm E, & Gallagher T. (2021). Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles. Cells, 10(4), 853.

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SARS-CoV-2 VLP Production and Characterization

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WT or MAGED2 knockout HEK293T cells in 10 cm dish were co-transfected with equal amounts of plasmids (24 µg in total) encoding the SARS-CoV-2 S, E, M, and HiBiT nucleocapsids using Vigofect transfection reagent (T001, Vigorous Biotechnology, China) following standard protocol. Supernatant containing VLPs were collected at 24-h post-transfection, and cell debris was removed by centrifugation at 4,000 rpm, 4°C for 30 min. VLPs in supernatant were then concentrated by 20% sucrose centrifugation at 100,000 × g, 4°C for 3 h. Pellets were dissolved in PBS and then separated by 10%–60% sucrose gradient centrifugation at 100,000 × g, 4°C for 3 h. VLPs in different fractions were measured by Nano-Glo luciferase assay reagents (N1110, Promega, CA, USA). Briefly, aliquots of each fraction were mixed with LgBiT protein and Nano-Glo luciferase assay substrate (Promega). Nluc activity was measured by GloMax Discover System (Promega).

Ju X., Wang Z., Wang P., Ren W., Yu Y., Yu Y., Yuan B., Song J., Zhang X., Zhang Y., Xu C., Tian B., Shi Y., Zhang R, & Ding Q. (2023). SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense. mBio, 14(4), e01373-23.

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SARS-CoV-2 Pseudovirus Neutralization Assay

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The pseudovirus neutralization assay was performed using Huh-7 cells stably expressing hACE2. The cells (100 μL, 3,000 in DMEM) were seeded on a 96-well plate overnight. Various concentrations of mAbs (4-fold serial dilutions starting at 30 μg/mL, 50 μL aliquots in triplicates) were mixed with the same volume of SARS-CoV-2 pseudovirus in a 96 well-plate. The mixture was incubated for 1 hour at 37°C with 5% CO₂. No-virus control wells were supplied with 100 μL DMEM medium (1% (v/v) Antibiotic-Antimycotic, 25 nM HEPES, 10% (v/v) FBS). Virus-only control wells contained 50 μL medium and 50 μL pseudovirus. After 1 h, medium was removed from Huh-7 cells, and then 100 μL pseudovirus and antibody mixture was incubated with the cells for 1 hour at 37°C with 5% CO₂. Another 100 μL DMEM medium was added into each well and incubated with the cells for 48 hours at 37°C with 5% CO₂. After the incubation, supernatants were removed, and 100 μL Nano-Glo® Luciferase Assay Reagent (Promega) (1:1 diluted in PBS) was added to each well and incubated for 5 min. Luminescence was measured using CLARIOstar Plus Microplate Reader (BMG labtech). The relative luciferase unit (RLU) was calculated by normalizing luminescence signal to the virus-only control group. IC50 was determined by a four-parameter nonlinear regression using GraphPad Prism 9.0 (GraphPad Software Inc.).

Fang Y., Sun P., Xie X., Du M., Du F., Ye J., Kalveram B.K., Plante J.A., Plante K.S., Li B., Bai X.C., Shi P.Y, & Chen Z.J. (2022). An antibody that neutralizes SARS-CoV-1 and SARS-CoV-2 by binding to a conserved spike epitope outside the receptor binding motif. Science Immunology.

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Quantifying Giardia Trophozoite Luminescence

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Giardia trophozoites were grown to confluency at 37°C for 48 h, iced for 15 min to detach trophozoites, and centrifuged at 900 × g for 5 min at 4°C. Trophozoites were resuspended in 1 ml of cold media, and serial dilutions (10^–1, 10^–2) were made for enumeration using a hemocytometer. To measure luminescence, three 50-µl aliquots of the 10^–2 dilution (∼1000 cells) were loaded into a white opaque 96-well assay plate (Corning Costar) and incubated for 30 min at 37°C. Following incubation, 50 µl of Nano-Glo Luciferase assay reagent (Promega), prepared at a 1:50 ratio of substrate to buffer, was added to each well. Luminescence was analyzed on a VictorX3 plate reader warmed to 37°C, using 0.1-s exposures, repeated every 30 s until maximal signal was detected. Experiments were performed using two independent samples with three technical replicates that were averaged over three different luminescence acquisitions and displayed with 95% confidence intervals. To determine the linear dynamic range of detection for the NanoLucNeo strain, we used 10-fold serial dilutions and plotted the luminescence readings versus the number of cells loaded into each well (Supplemental Material).

McInally S.G., Hagen K.D., Nosala C., Williams J., Nguyen K., Booker J., Jones K, & Dawson S.C. (2019). Robust and stable transcriptional repression in Giardia using CRISPRi. Molecular Biology of the Cell, 30(1), 119-130.

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SARS-CoV-2 Pseudovirus Neutralization Assay

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The pseudovirus neutralization assays were performed using ACE2-Expressing Huh-7. Huh-7 cells (100 μL, 5 × 10³ in DMEM) were added to 96 well-plate for overnight incubation. Various concentrations of mAbs (4-fold serial dilution with starting point at 30 μg/mL, 50 μL aliquots, triplicates) were mixed with the same volume of SARS-CoV-2 pseudovirus in a 96 well-plate. The mixture was incubated for 1 h at 37 °C, supplied with 5% CO₂. No virus control wells were supplied with 100 μL DMEM (1% (v/v) antibiotics, 25 nM HEPES, 10% (v/v) FBS). Virus only control wells were supplied with 50 μL DMEM and 50 μL pseudovirus. After 1 h, medium was removed from Huh-7 cells, and then 100 μL pseudovirus and antibody mixture were added into Huh-7 cells containing plates. The 96-well plates were incubated for 48 h at 37 °C supplied with 5% CO₂. After the incubation, supernatants were removed, and 100 μL Nano-Glo® Luciferase Assay Reagent (Promega) (1:1 diluted in PBS) was added to each well and incubated for 5 mins. After the incubation, the Luminescence was measured using CLARIOstar Plus Microplate Reader (BMG labtech). The relative luciferase unit (RLU) was calculated by normalizing Luminescence signal to virus only control group. IC₅₀ were determined by a four-parameter nonlinear regression using GraphPad Prism 9.0 (GraphPad Software Inc.).

Beshnova D., Fang Y., Du M., Sun Y., Du F., Ye J., Chen Z.J, & Li B. (2022). Computational approach for binding prediction of SARS-CoV-2 with neutralizing antibodies. Computational and Structural Biotechnology Journal, 20, 2212-2222.

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Luminescence-based Transfection Assay

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Candidate RNAs were serially diluted, complexed with lipofectamine 2000 using the manufacturer’s instructions, and added to white 96-well plates. BHK cells then were added to each well, and 16 to 24 h after transfection, Nano-Glo Luciferase Assay Reagent (Promega) was added to each well, and luminescence was measured.

Erasmus J.H., Archer J., Fuerte-Stone J., Khandhar A.P., Voigt E., Granger B., Bombardi R.G., Govero J., Tan Q., Durnell L.A., Coler R.N., Diamond M.S., Crowe JE J.r., Reed S.G., Thackray L.B., Carnahan R.H, & Van Hoeven N. (2020). Intramuscular Delivery of Replicon RNA Encoding ZIKV-117 Human Monoclonal Antibody Protects against Zika Virus Infection. Molecular Therapy. Methods & Clinical Development, 18, 402-414.

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Measuring Autoantibodies Against IFN-α Subtypes

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Levels of autoantibodies against IFN-α subtypes were measured in luciferase-based immunoprecipitation assay (LIPS), as previously described (9). IFNA1, IFNA2, IFNA8, and IFNA21 sequences were inserted into a modified pPK-CMV-F4 fusion vector (PromoCell GmbH, Germany), in which the firefly luciferase replaced the NanoLuc luciferase (Promega, USA). The resulting constructs were used to transfect HEK293 cells and the IFNA-luciferase fusion proteins were collected in the tissue culture supernatant. For autoantibody screening, we combined 2x10⁶ luminescence units (LU) of IFNA1, IFNA2, IFNA8 and IFNA21 in a single IP reaction mixture (pool 1), and IFNA4, IFNA5, IFNA6 and IFNA7 in another IP reaction mixture (pool 2). Serum samples were incubated with Protein G agarose beads (Exalpha Biologicals, USA) at room temperature for 1 hour in a 96-well microfilter plate (Merck Millipore, Germany), and we then added 2x10⁶ luminescence units (LU) of antigen and incubated for another hour. Each sample was tested once. The plate was washed with a vacuum system and Nano-Glo® Luciferase Assay Reagent (Promega, USA) was added. Luminescence intensity was measured with a VICTOR X Multilabel Plate Reader (PerkinElmer Life Sciences, USA). The results are expressed in arbitrary units (AU), as a fold-difference relative to the mean of the negative control samples.

Bastard P., Gervais A., Le Voyer T., Rosain J., Philippot Q., Manry J., Michailidis E., Hoffmann H.H., Eto S., Garcia-Prat M., Bizien L., Parra-Martínez A., Yang R., Haljasmägi L., Migaud M., Särekannu K., Maslovskaja J., de Prost N., Tandjaoui-Lambiotte Y., Luyt C.E., Amador-Borrero B., Gaudet A., Poissy J., Morel P., Richard P., Cognasse F., Troya J., Trouillet-Assant S., Belot A., Saker K., Garçon P., Rivière J.G., Lagier J.C., Gentile S., Rosen L.B., Shaw E., Morio T., Tanaka J., Dalmau D., Tharaux P.L., Sene D., Stepanian A., Megarbane B., Triantafyllia V., Fekkar A., Heath J.R., Franco J.L., Anaya J.M., Solé-Violán J., Imberti L., Biondi A., Bonfanti P., Castagnoli R., Delmonte O.M., Zhang Y., Snow A.L., Holland S.M., Biggs C., Moncada-Vélez M., Arias A.A., Lorenzo L., Boucherit S., Coulibaly B., Anglicheau D., Planas A.M., Haerynck F., Duvlis S., Nussbaum R.L., Ozcelik T., Keles S., Bousfiha A.A., El Bakkouri J., Ramirez-Santana C., Paul S., Pan-Hammarström Q., Hammarström L., Dupont A., Kurolap A., Metz C.N., Aiuti A., Casari G., Lampasona V., Ciceri F., Barreiros L.A., Dominguez-Garrido E., Vidigal M., Zatz M., van de Beek D., Sahanic S., Tancevski I., Stepanovskyy Y., Boyarchuk O., Nukui Y., Tsumura M., Vidaur L., Tangye S.G., Burrel S., Duffy D., Quintana-Murci L., Klocperk A., Kann N.Y., Shcherbina A., Lau Y.L., Leung D., Coulongeat M., Marlet J., Koning R., Reyes L.F., Chauvineau-Grenier A., Venet F., Monneret G., Nussenzweig M.C., Arrestier R., Boudhabhay I., Baris-Feldman H., Hagin D., Wauters J., Meyts I., Dyer A.H., Kennelly S.P., Bourke N.M., Halwani R., Sharif-Askari N.S., Dorgham K., Sallette J., Sedkaoui S.M., AlKhater S., Rigo-Bonnin R., Morandeira F., Roussel L., Vinh D.C., Ostrowski S.R., Condino-Neto A., Prando C., Bonradenko A., Spaan A.N., Gilardin L., Fellay J., Lyonnet S., Bilguvar K., Lifton R.P., Mane S., Anderson M.S., Boisson B., Béziat V., Zhang S.Y., Vandreakos E., Hermine O., Pujol A., Peterson P., Mogensen T.H., Rowen L., Mond J., Debette S., de Lamballerie X., Duval X., Mentré F., Zins M., Soler-Palacin P., Colobran R., Gorochov G., Solanich X., Susen S., Martinez-Picado J., Raoult D., Vasse M., Gregersen P.K., Piemonti L., Rodríguez-Gallego C., Notarangelo L.D., Su H.C., Kisand K., Okada S., Puel A., Jouanguy E., Rice C.M., Tiberghien P., Zhang Q., Cobat A., Abel L., Casanova J.L., Bigio B., Boucherit S., de la Chapelle A., Chen J., Chrabieh M., Coulibaly B., Liu D., Nemirowskaya Y., Cruz I.M., Materna M., Pelet S., Seeleuthner Y., Thibault C., Liu Z., Abad J., Accordino G., Achille C., Aguilera-Albesa S., Aguiló-Cucurull A., Aiuti A., Özkan E.A., Darazam I.A., Roblero Albisures J.A., Aldave J.C., Ramos M.A., Khan T.A., Aliberti A., Nadji S.A., Alkan G., Alkhater S.A., Allardet-Servent J., Allende L.M., Alonso-Arias R., Alshahrani M.S., Alsina L., Alyanakian M.A., Borrero B.A., Amoura Z., Antolí A., Arrestier R., Aubart M., Auguet T., Avramenko I., Aytekin G., Azot A., Bahram S., Bajolle F., Baldanti F., Baldolli A., Ballester M., Feldman H.B., Barrou B., Barzagh F., Basso S., Bayhan G.I., Belot A., Bezrodnik L., Bilbao A., Blanchard-Rohner G., Blanco I., Blandinières A., Blázquez-Gamero D., Bleibtreu A., Bloomfield M., Bolivar-Prados M., Bondarenko A., Borghesi A., Borie R., Botdhlo-Nevers E., Bousfiha A.A., Bousquet A., Boutolleau D., Bouvattier C., Boyarchuk O., Bravais J., Briones M.L., Brunner M.E., Bruno R., Bueno M.R., Bukhari H., Bustamante J., Cáceres Agra J.J., Capra R., Carapito R., Carrabba M., Casari G., Casasnovas C., Caseris M., Cassaniti I., Castelle M., Castelli F., de Vera M.C., Castro M.V., Catherinot E., Celik J.B., Ceschi A., Chalumeau M., Charbit B., Cheng M.P., Clavé P., Clotet B., Codina A., Cohen Y., Colobran R., Comarmond C., Combes A., Comoli P., Corsico A.G., Coşkuner T., Cvetkovski A., Cyrus C., Dalmau D., Danion F., Darley D.R., Das V., Dauby N., Dauger S., De Munter P., de Pontual L., Dehban A., Delplancq G., Demoule A., Desguerre I., Di Sabatino A., Diehl J.L., Dobbelaere S., Domínguez-Garrido E., Dubost C., Ekwall O., Bozdemir Ş.E., Elnagdy M.H., Emiroglu M., Endo A., Erdeniz E.H., Aytekin S.E., Lasa M.P., Euvrard R., Fabio G., Faivre L., Falck A., Fartoukh M., Faure M., Arquero M.F., Ferrer R., Ferreres J., Flores C., Francois B., Fumadó V., Fung K.S., Fusco F., Gagro A., Solis B.G., Gaussem P., Gayretli Z., Gil-Herrera J., Gilardin L., Gatineau A.G., Girona-Alarcón M., Cifuentes Godínez K.A., Goffard J.C., Gonzales N., Gonzalez-Granado L.I., González-Montelongo R., Guerder A., Gülhan B., Gumucio V.D., Hanitsch L.G., Gunst J., Gut M., Hadjadj J., Haerynck F., Halwani R., Hammarström L., Hancerli S., Hariyan T., Hatipoglu N., Heppekcan D., Hernandez-Brito E., Ho P.K., Holanda-Peña M.S., Horcajada J.P., Hraiech S., Humbert L., Hung I.F., Iglesias A.D., Íñigo-Campos A., Jamme M., Arranz M.J., Jimeno M.T., Jordan I., Yüksek S.K., Kara Y.B., Karahan A., Karbuz A., Yasar K.K., Kasapcopur O., Kashimada K., Keles S., Demirkol Y.K., Kido Y., Kizil C., Kılıç A.O., Klocperk A., Koutsoukou A., Król Z.J., Ksouri H., Kuentz P., Kwan A.M., Kwan Y.W., Kwok J.S., Lagier J.C., Lam D.S., Lampropoulou V., Lanternier F., Lau Y.L., Le Bourgeois F., Leo Y.S., Lopez R.L., Leung D., Levin M., Levy M., Lévy R., Li Z., Lilleri D., Lima E.J., Linglart A., López-Collazo E., Lorenzo-Salazar J.M., Louapre C., Lubetzki C., Lung K.C., Luyt C.E., Lye D.C., Magnone C., Mansouri D., Marchioni E., Marioli C., Marjani M., Marques L., Pereira J.M., Martín-Nalda A., Pueyo D.M., Martinez-Picado J., Marzana I., Mata-Martínez C., Mathian A., Matos L.R., Matthews G.V., Mayaux J., McLaughlin-Garcia R., Meersseman P., Mège J.L., Mekontso-Dessap A., Melki I., Meloni F., Meritet J.F., Merlani P., Akcan Ö.M., Meyts I., Mezidi M., Migeotte I., Millereux M., Million M., Mirault T., Mircher C., Mirsaeidi M., Mizoguchi Y., Modi B.P., Mojoli F., Moncomble E., Melián A.M., Martinez A.M., Morandeira F., Morange P.E., Mordacq C., Morelle G., Mouly S.J., Muñoz-Barrera A., Nafati C., Nagashima S., Nakagama Y., Neven B., Neves J.F., Ng L.F., Ng Y.Y., Nielly H., Medina Y.N., Cuadros E.N., Ocejo-Vinyals J.G., Okamoto K., Oualha M., Ouedrani A., Özçelik T., Ozkaya-Parlakay A., Pagani M., Pan-Hammarström Q., Papadaki M., Parizot C., Parola P., Pascreau T., Paul S., Paz-Artal E., Pedraza S., González Pellecer N.C., Pellegrini S., de Diego R.P., Pérez-Fernández X.L., Philippe A., Philippot Q., Picod A., de Chambrun M.P., Piralla A., Planas-Serra L., Ploin D., Poissy J., Poncelet G., Poulakou G., Pouletty M.S., Pourshahnazari P., Qiu-Chen J.L., Quentric P., Rambaud T., Raoult D., Raoult V., Rebillat A.S., Redin C., Resmini L., Ricart P., Richard J.C., Rigo-Bonnin R., Rivet N., Rivière J.G., Rocamora-Blanch G., Rodero M.P., Rodrigo C., Rodriguez L.A., Rodriguez-Gallego C., Rodriguez-Palmero A., Romero C.S., Rothenbuhler A., Roux D., Rovina N., Rozenberg F., Ruch Y., Ruiz M., Ruiz del Prado M.Y., Ruiz-Rodriguez J.C., Sabater-Riera J., Saks K., Salagianni M., Sanchez O., Sánchez-Montalvá A., Sánchez-Ramón S., Schidlowski L., Schluter A., Schmidt J., Schmidt M., Schuetz C., Schweitzer C.E., Scolari F., Sediva A., Seijo L., Seminario A.G., Sene D., Seng P., Senoglu S., Seppänen M., Llovich A.S., Shahrooei M., Shcherbina A., Siguret V., Siouti E., Smadja D.M., Smith N., Sobh A., Solanich X., Solé-Violán J., Soler C., Soler-Palacín P., Sözeri B., Stella G.M., Stepanovskiy Y., Stoclin A., Taccone F., Tandjaoui-Lambiotte Y., Taupin J.L., Tavernier S.J., Tello L.V., Terrier B., Thiery G., Thorball C., THORN K., Thumerelle C., Tipu I., Tolstrup M., Tomasoni G., Toubiana J., Alvarez J.T., Triantafyllia V., Trouillet-Assant S., Troya J., Tsang O.T., Tserel L., Tso E.Y., Tucci A., Tüter Öz Ş.K., Ursini M.V., Utsumi T., Uzunhan Y., Vabres P., Valencia-Ramos J., Van Den Rym A.M., Vandernoot I., Velez-Santamaria V., Zuniga Veliz S.P., Vidigal M.C., Viel S., Vilain C., Vilaire-Meunier M.E., Villar-García J., Vincent A., Vogt G., Voiriot G., Volokha A., Vuotto F., Wauters E., Wauters J., Wu A.K., Wu T.C., Yahşi A., Yesilbas O., Yildiz M., Young B.E., Yükselmiş U., Zatz M., Zecca M., Zuccaro V., Jens V.P., Lambrecht B.N., Eva V.B., Cédric B., Levi H., Eric H., Bauters F., De Clercq J., Cathérine H., Hans S., Leslie N., Florkin B., Boulanger C., Vanderlinden D., Foti G., Bellani G., Citerio G., Contro E., Pesci A., Valsecchi M.G., Cazzaniga M., Danielson J.J., Dobbs K., Kashyap A., Ding L., Dalgard C.L., Sottini A., Quaresima V., Quiros-Roldan E., Rossi C., Bettini L.R., D’Angio’ M., Beretta I., Montagna D., Licari A., Marseglia G.L., Batten I., Reddy C., McElheron M., Noonan C., Connolly E., Fallon A., Storgaard M., Jørgensen S., Tolstrup M., Erikstrup C., Pedersen O.B., Sørensen E., Mikkelsen S., Dinh K.M., Larsen M.A., Paulsen I.W., Von Stemann J.H., Hansen M.B., Ostrowski S.R., Townsend L., Cheallaigh C.N., Bergin C., Martin-Loeches I., Dunne J., Conlon N., Bourke N., O'Farrelly C., Abel L., Allavena C., Andrejak C., Angoulvant F., Azoulay C., Bachelet D., Bartoli M., Basmaci R., Behilill S., Beluze M., Benech N., Benkerrou D., Bhavsar K., Bitker L., Bouadma L., Bouscambert-Duchamp M., Paz P.C., Cervantes-Gonzalez M., Chair A., Chirouze C., Coelho A., Cordel H., Couffignal C., Couffin-Cadiergues S., d’Ortenzio E., De Montmollin E., Debard A., Debray M.P., Deplanque D., Descamps D., Desvallée M., Diallo A., Diehl J.L., Diouf A., Dorival C., Dubos F., Duval X., Eloy P., Enouf V., Epaulard O., Esperou H., Esposito-Farese M., Etienne M., Garot D., Gault N., Gaymard A., Ghosn J., Gigante T., Gilg M., Goehringer F., Guedj J., Hoctin A., Hoffmann I., Houas I., Hulot J.S., Jaafoura S., Kafif O., Kaguelidou F., Kali S., Kerroumi Y., Khalil A., Khan C., Kimmoun A., Laine F., Laouénan C., Laribi S., Le M., Le Bris C., Le Gac S., Le Hingrat Q., Le Mestre S., Le Nagard H., Lemaignen A., Lemee V., Lescure F.X., Letrou S., Levy Y., Lina B., Lingas G., Lucet J.C., Machado M., Malvy D., Mambert M., Manuel A., Mentré F., Meziane A., Mouquet H., Mullaert J., Neant N., Nguyen D., Noret M., Papadopoulos A., Paul C., Peiffer-Smadja N., Peigne V., Petrov-Sanchez V., Peytavin G., Pham H., Picone O., Piquard V., Poissy J., Puéchal O., Rosa-Calatrava M., Rossignol B., Rossignol P., Roy C., Schneider M., Su R., Tardivon C., Tellier M.C., Téoulé F., Terrier O., Timsit J.F., Tual C., Tubiana S., Van Der Werf S., Vanel N., Veislinger A., Visseaux B., Wiedemann A., Yazdanpanah Y., Annereau J.P., Briseño-Roa L., Gribouval O., Pelet A., Abel L., Alcover A., Aschard H., Bousso P., Bourke N., Brodin P., Bruhns P., Cerf-Bensussan N., Cumano A., D’Enfert C., Deriano L., Dillies M.A., Di Santo J., Dromer F., Eberl G., Enninga J., Fellay J., Gomperts-Boneca I., Hasan M., Hedestam G.K., Hercberg S., Ingersoll M.A., Lantz O., Kenny R.A., Ménager M., Michel F., Mouquet H., O'Farrelly C., Patin E., Pellegrini S., Rausell A., Rieux-Laucat F., Rogge L., Fontes M., Sakuntabhai A., Schwartz O., Schwikowski B., Shorte S., Tangy F., Toubert A., Touvier M., Ungeheuer M.N., Zimmer C., Albert M.L., Duffy D., Quintana-Murci L., Alavoine L., Behillil S., Burdet C., Charpentier C., Dechanet A., Descamps D., Duval X., Ecobichon J.L., Enouf V., Frezouls W., Houhou N., Kafif O., Lehacaut J., Letrou S., Lina B., Lucet J.C., Manchon P., Nouroudine M., Piquard V., Quintin C., Thy M., Tubiana S., van der Werf S., Vignali V., Visseaux B., Yazdanpanah Y., Chahine A., Waucquier N., Migaud M.C., Deplanque D., Djossou F., Mergeay-Fabre M., Lucarelli A., Demar M., Bruneau L., Gérardin P., Maillot A., Payet C., Laviolle B., Laine F., Paris C., Desille-Dugast M., Fouchard J., Malvy D., Nguyen D., Pistone T., Perreau P., Gissot V., Le Goas C., Montagne S., Richard L., Chirouze C., Bouiller K., Desmarets M., Meunier A., Lefévre B., Jeulin H., Legrand K., Lomazzi S., Tardy B., Gagneux-Brunon A., Bertholon F., Botelho-Nevers E., Kouakam C., Leturque N., Roufai L., Amat K., Couffin-Cadiergues S., Espérou H., Hendou S., van 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Vlaar A.P., van de Beek D., Abel L., Aiuti A., Al-Muhsen S., Al-Mulla F., Anderson M.S., Andreakos E., Arias A.A., Feldman H.B., Belot A., Biggs C.M., Bogunovic D., Bolze A., Bondarenko A., Bousfiha A.A., Brodin P., Bryceson Y., Bustamante C.D., Butte M.J., Casari G., Chakravorty S., Christodoulou J., Condino-Neto A., Constantinescu S.N., Cooper M.A., Dalgard C.L., Desai M., Drolet B.A., El Baghdadi J., Espinosa-Padilla S., Fellay J., Flores C., Franco J.L., Froidure A., Gregersen P.K., Haerynck F., Hagin D., Halwani R., Hammarström L., Heath J.R., Henrickson S.E., Hsieh E.W., Husebye E.S., Imai K., Itan Y., Jarvis E.D., Karamitros T., Kisand K., Ku C.L., Lau Y.L., Ling Y., Lucas C.L., Maniatis T., Mansouri D., Maródi L., Meyts I., Milner J.D., Mironska K., Mogensen T.H., Morio T., Ng L.F., Notarangelo L.D., Novelli A., Novelli G., O'Farrelly C., Okada S., Ozcelik T., Pan-Hammarström Q., de Diego R.P., Planas A.M., Prando C., Pujol A., Quintana-Murci L., Renia L., Resnick I., Rodríguez-Gallego C., Sancho-Shimizu V., Sediva A., Seppänen M.R., Shahrooei M., Shcherbina A., Slaby O., Snow A.L., Soler-Palacín P., Spaan A.N., Tancevski I., Tangye S.G., Abou Tayoun A., Ramaswamy S., Turvey S.E., Uddin K.M., Uddin M.J., van de Beek D., Vinh D.C., von Bernuth H., Zatz M., Zawadzki P., Su H.C., Casanova J.L., Nadif R., Goldberg M., Ozguler A., Henny J., Lemonnier S., Coeuret-Pellicer M., Le Got S., Zins M., Tzourio C., Debette S., Dufouil C., Soumaré A., Lachaize M., Fievet N., Flaig A., Martin F., Bonneaudeau B., Cannet D., Gallian P., Jeanne M., Perroquin M, & Hamzeh-Cognasse H. (2021). Autoantibodies neutralizing type I IFNs are present in ~4% of uninfected individuals over 70 years old and account for ~20% of COVID-19 deaths. Science Immunology, 6(62), eabl4340.

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Luciferase Assay for SPIKE Particle Analysis

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For luciferase assays, cells were grown in 96-well, white tissue-culture plates (Corning). After exposure to SPIKE particles as indicated, cells were lysed with Nano-Glo Luciferase Assay reagent (Promega). Luciferase reagent was prepared according to the manufacturer’s protocol. For lysis, 100 μl of a 1:1 mix of luciferase reagent and cell culture media was added to each well of the 96-well plate and incubated at room temperature for 3 minutes. The plate was read using a Biotek Synergy H1 multimode plate reader.

Bednash J.S., Johns F., Farkas D., Elhance A., Adair J., Cress K., Yount J.S., Kenney A.D., Londino J.D, & Mallampalli R.K. (2023). Inhibiting the Deubiquitinase UCHL1 Reduces SARS-CoV-2 Viral Uptake by ACE2. American Journal of Respiratory Cell and Molecular Biology, 68(5), 566-576.

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Nanoluciferase Assay in HeLa Cells

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HeLa cells were seeded into six-well plates at a density of 2 × 10⁵ cells/well. The cells were transfected with Lipofectamine 3000 reagent (Invitrogen), according to the manufacturer’s protocol. The cells were harvested 1 to 6 days after transfection, and the nanoluciferase activity in the cells was measured using the Nano-Glo luciferase assay reagent (Promega).

Monde K., Satou Y., Goto M., Uchiyama Y., Ito J., Kaitsuka T., Terasawa H., Monde N., Yamaga S., Matsusako T., Wei F.Y., Inoue I., Tomizawa K., Ono A., Era T., Sawa T, & Maeda Y. (2022). Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells. Journal of Virology, 96(9), e00356-22.

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ACE-tRNA PTC Readthrough Assay

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The day before transfection, HEK293 cells (ATCC, USA) (<40 passages) were plated at 1.4 × 10⁴ cells/well in 96-well cell culture treated plates in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% FBS, 1% Pen/Step and 2 mM l-Glutamine (Thermofisher, USA). The all-in-one nonsense reporter with ACE-tRNA genes were transfected in triplicate/plate using Calfectin (Signagen, USA). Sixteen hours post-transfection, the media was aspirated and 20 µl of PBS was added to each well. Fifteen microliter of lytic Nano-Glo® Luciferase Assay Reagent was added to each well (1:50 reagent to buffer; Promega, USA). The plates were incubated for 2 min after rotational shaking and read using a SpectraMax i3 plate reader (Molecular Devices, USA; integration time, 200 ms; All wavelengths collected in endpoint mode). Luminescence was averaged across three wells for each experiment and all ACE-tRNAs were repeated >3 times in this fashion. Each plate also contained in triplicate wells transfected with the all-in-one nonsense reporter with no ACE-tRNA to server as control for transfection efficiency and baseline PTC readthrough. All values are reported as ratios of ACE-tRNA luminescence over baseline PTC readthrough luminescence ± SEM. One-way ANOVAs were performed with Tukey’s post-hoc analysis across all ACE-tRNAs in a given amino acid family, Supplementary Data 2.

Lueck J.D., Yoon J.S., Perales-Puchalt A., Mackey A.L., Infield D.T., Behlke M.A., Pope M.R., Weiner D.B., Skach W.R., McCray PB J.r, & Ahern C.A. (2019). Engineered transfer RNAs for suppression of premature termination codons. Nature Communications, 10, 822.

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