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Luciferase reporter assay

TA Takaki Asano
BB Bertrand Boisson
JC Jean-Laurent Casanova

Last updated date: May 7, 2024 Views: 558 Forks: 0

original An abbreviated version of this protocol was published in Science Immunology in Aug, 2021
X-linked recessive TLR7 deficiency in ~1% of men under 60 years old with life-threatening COVID-19
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TLR7 LUC

Day1: Plating 

HEK293T cells (TLR7/8 null): 2.5x10^4 cells/well (96 well plate) 

 

Day2: Transfection

Transfection using X-treamGene9 (OptiMEM : X-tremeGene9 : DNA = 100ul : 3ul : 1ug)

NF-kB reporter (pGL4.32) (Firefly): 100ng

The pGL4.32 (luc2P/NF-κB–RE/Hygo) (Promega) reporter vector contains five copies of the

NF-κB–responsive element (NF-κB–RE) linked to the luciferase reporter gene luc2P.

pRLTK (Renilla): 10ng

TLR7, or EV (pCMV6-Kan): 50ng

(15min RT, and then transfer them to each well)

 

Day3: Stimulation.

Optional : Change media. (extra-cautious since cell can detach)

For TLR7: R848 1ug/ml (TLR7/8 agonist), CL264 5 ug/ml (TLR7 agonist), Imiquimod 5 ug/ml (TLR7 agonist)

 

Day4: Measure LUC

Dual-Gro Stop & Glo Reagent

  1. remove 40ul media
  2. Dual-Glo Luciferase Reagent 60ul/well (no bubble)
  3. 10-15 min / RT (shaking in dark) 
  4. <Firefly> measure 556nm fluorescence
  5. Dual-Glo Stop & Glo reagent 60ul/well (no bubble)
  6. 10-15 min / RT (shaking in dark)
  7. <Renilla> measure 480nm fluorescence
Copyright: © 2024 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Asano, T, Boisson, B and Casanova, J(2024). Luciferase reporter assay. Bio-protocol Preprint. bio-protocol.org/prep2666.
  2. Asano, T., Boisson, B., Onodi, F., Matuozzo, D., Moncada-Velez, M., Renkilaraj, M. R. L. M., Zhang, P., Meertens, L., Bolze, A., Materna, M., Korniotis, S., Gervais, A., Talouarn, E., Bigio, B., Seeleuthner, Y., Bilguvar, K., Zhang, Y., Neehus, A., Ogishi, M., Pelham, S. J., Voyer, T. L., Rosain, J., Philippot, Q., Soler-Palacín, P., Colobran, R., Martin-Nalda, A., Rivière, J. G., Tandjaoui-Lambiotte, Y., Chaïbi, K., Shahrooei, M., Darazam, I. A., Olyaei, N. A., Mansouri, D., Hatipoğlu, N., Palabiyik, F., Ozcelik, T., Novelli, G., Novelli, A., Casari, G., Aiuti, A., Carrera, P., Bondesan, S., Barzaghi, F., Rovere-Querini, P., Tresoldi, C., Franco, J. L., Rojas, J., Reyes, L. F., Bustos, I. G., Arias, A. A., Morelle, G., Kyheng, C., Troya, J., Planas-Serra, L., Schlüter, A., Gut, M., Pujol, A., Allende, L. M., Rodriguez-Gallego, C., Flores, C., Cabrera-Marante, O., Pleguezuelo, D. E., Diego, R. P. D., Keles, S., Aytekin, G., Akcan, O. M., Bryceson, Y. T., Bergman, P., Brodin, P., Smole, D., Smith, C. I. E., Norlin, A., Campbell, T. M., Covill, L. E., Hammarström, L., Pan-Hammarström, Q., Abolhassani, H., Mane, S., Marr, N., Ata, M., Ali, F. A., Khan, T., Spaan, A. N., Dalgard, C. L., Bonfanti, P., Biondi, A., Tubiana, S., Burdet, C., Nussbaum, R., Kahn-Kirby, A., Snow, A. L., Bustamante, J., Puel, A., Boisson-Dupuis, S., Zhang, S., Béziat, V., Lifton, R. P., Bastard, P., Notarangelo, L. D., Abel, L., Su, H. C., Jouanguy, E., Amara, A., Soumelis, V., Cobat, A., Zhang, Q. and Casanova, J.(2021). X-linked recessive TLR7 deficiency in ~1% of men under 60 years old with life-threatening COVID-19. Science Immunology 6(62). DOI: 10.1126/sciimmunol.abl4348

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