Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle

Kata Tombácz*, Duncan Mwangi, Dirk Werling, Amanda J. Gibson

*Awdur cyfatebol y gwaith hwn

Allbwn ymchwil: Cyfraniad at gyfnodolynErthygladolygiad gan gymheiriaid

6 Dyfyniadau (Scopus)
71 Wedi eu Llwytho i Lawr (Pure)

Crynodeb

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-B-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-B reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

Iaith wreiddiolSaesneg
Tudalennau (o-i)329-335
Nifer y tudalennau7
CyfnodolynInnate Immunity
Cyfrol23
Rhif cyhoeddi4
Dyddiad ar-lein cynnar27 Chwef 2017
Dynodwyr Gwrthrych Digidol (DOIs)
StatwsCyhoeddwyd - 01 Mai 2017

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