Protocol for evaluation and validation of TLR8 antagonists in HEK-Blue cells via secreted embryonic alkaline phosphatase assay

Zhenyi Hu; Tuan Zhang; Shuangshuang Jiang; Hang Yin

doi:10.1016/j.xpro.2021.101061

Protocol for evaluation and validation of TLR8 antagonists in HEK-Blue cells via secreted embryonic alkaline phosphatase assay

STAR Protoc. 2021 Dec 23;3(1):101061. doi: 10.1016/j.xpro.2021.101061. eCollection 2022 Mar 18.

Authors

Zhenyi Hu¹, Tuan Zhang^{1

2

3}, Shuangshuang Jiang^{1

2

3}, Hang Yin^{1

2

3}

Affiliations

¹ School of Pharmaceutical Sciences, Tsinghua University, 100084 Beijing, China.
² Tsinghua-Peking Center for Life Sciences, Tsinghua University, 100084 Beijing, China.
³ Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, 100084 Beijing, China.

Abstract

Toll-like receptor 8 (TLR8) is a pattern recognition receptor that senses RNA degradation products and initiates immune responses. TLR8 overactivation is associated with autoimmune diseases. Herein, we describe the evaluation and validation of TLR8 antagonists in HEK-Blue cells via secreted embryonic alkaline phosphatase (SEAP) assay, WST assay, ITC and immunoblotting. These assays can facilitate the development of TLR8 antagonists; this protocol can also be adapted to analyze agonists and antagonists for other TLRs. For complete details on the use and execution of this protocol, please refer to Hu et al. (2018).

Keywords: Cell culture; Cell-based Assays; Chemistry; High Throughput Screening; Molecular/Chemical Probes.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alkaline Phosphatase*
Cell Line
Humans
Toll-Like Receptor 8* / antagonists & inhibitors

Substances

Toll-Like Receptor 8
Alkaline Phosphatase