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

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.

1. Identify the optimal LPS dose by generating a dose-response curve prior to the screening.​
   1. Dilute the working LPS solution to a final concentration of 0.01 - ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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