An Assay to Screen Bioactive Nanoparticles for Toll-Like Receptor Signaling Inhibition

Published: January 31, 2024

Abstract

Source: Yang, H., et al. Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling. J. Vis. Exp. (2017).

This video demonstrates a reporter cell-based assay for screening potential bioactive nanoparticles that inhibit Toll-like receptor (TLR) signaling. The assay involves introducing lipopolysaccharides (LPS) mixed with peptide-gold nanoparticle hybrids to reporter macrophages expressing the reporter proteins — secreted embryonic alkaline phosphatase (SEAP) and luciferase. The reduction of LPS-induced TLR signal mediated by the hybrids is determined by measuring the reporter signals.

Protocol

1. Screening for Potential TLR4 Nano-inhibitors Using the Reporter Cells NOTE: Since TLR4 signaling utilizes both MyD88-dependent land TRIF-dependent pathways, it is selected as the primary target to encompass a wide range of TLR signaling pathways. THP-1-XBlue reporter cells are used to mainly examine the NF-κB/AP-1 activation while THP-1-Dual cells are for IRF activation from the TRIF-dependent signal transduction. Identify the optimal LPS d…

Divulgations

The authors have nothing to disclose.

Materials

THP-1-XBlue reporter cell InvivoGen thpx-sp keep cell culture passage under 20
THP-1-Dual repoter cell InvivoGen thpd-nfis keep cell culture passage under 20
RPMI-1640 (no L-glutamine) GE Health Care SH30096.02 Warm up to 37 °C before use; add supplements to make a complete medium R10
Fetal bovine serum (qualified) Thermo Fisher Scientific 12484028 Heat inactivated; 10% in RPMI-1640
L-glutamine Thermo Fisher Scientific SH30034.02 2 mM in the complete medium R10
Sodium pyruvate Thermo Fisher Scientific 11360-070 1 mM in the complete medium R10
Dulbecco's phosphate buffered saline, 1X, without calcium, magnesium GE Health Care SH30028.02 Use for cell washing and reagent preparation
QUANTI-Blue InvivoGen rep-qb1 SEAP substrate
QUANTI-Luc InvivoGen rep-qlc2 Luciferase substrate
Zeocin InvivoGen ant-zn-1 Selection antibiotics for reporter cells
Blasticidin InvivoGen anti-bl-1 Selection antibiotics for reporter cells
Dimethyl sulfoxide (DMSO) for molecular biology Sigmal-Aldrich D8418-100ML Use for reagent preparation
Lipopolysaccharide (LPS) from E. coli K12 InvivoGen tlrl-eklps TLR4 ligand
Flagellin from S. Typhimurium (FLA-ST), ultrapure InvivoGen tlrl-epstfla TLR5 ligand
SpectraMax Plus 384 microplate reader Molecular Devices N/A Read colorimetric assay
Infinite M200 Pro multimode microplate reader with injectors Tecan N/A Read luminiscience
Costar assay plate, 96-well white with clear flat bottom, tissue culure treated Corning Costar 3903 Used for luminiscence assay

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Citer Cet Article
An Assay to Screen Bioactive Nanoparticles for Toll-Like Receptor Signaling Inhibition. J. Vis. Exp. (Pending Publication), e21928, doi: (2024).

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