An Assay for TLR-Dependent NF-кB/AP-1 Transcription Factor Signaling in Macrophages

Abstract

Source: McKiel, L. A., et al. A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces. J. Vis. Exp. (2020).

This video demonstrates an assay to examine the toll-like receptor(TLR)-dependent activity of the transcription factors nuclear factor-kappa-B (NF-κB) and activator protein-1 (AP-1) in macrophages. The TLRs trigger a pro-inflammatory signaling cascade upon recognizing damage-associated molecular patterns (DAMPs), activating NF-κB and AP-1. The engineered reporter macrophages produce secreted embryonic alkaline phosphatase (SEAP) enzyme upon NF-κB and AP-1 activation, which is detected via a chromogenic reaction.

Protocol

1. Media and Reagent Preparation Prepare fibroblast media. Combine 450 mL of Dulbecco's modified Eagle medium (DMEM), 50 mL of fetal bovine serum (FBS), and 5 mL of penicillin/streptomycin. Store at 4 °C for up to 3 months. Prepare reporter macrophage growth media in 50 mL aliquots. Combine 45 mL of DMEM, 5 mL of FBS, 5 μg/mL mycoplasma elimination reagent (Table of Materials), and 200 μg/mL phleomycin D1 (Table of Materials). Sto…

Representative Results

Figure 1: Methods and results for the alkaline phosphatase assay of NF-кB/AP-1 reporter macrophages on TCPS, PMMA, PDMS, and fPTFE. (A) Diagram of the workflow for the reporter macrophage alkaline phosphatase assay. (B) Western blot of lysate confirming the presence of DAMP species HMGB1 and HSP60, with β-actin as the loading control. (C</stron…

Materials

Cell culture reagents
anti-mouse/human CD282 (TLR2)	Biolegend	121802
CLI-095 (TLR4 inhibitor)	Invivogen	TLRL-CLI95
C57 complement plasma K2 EDTA 10ml, innovative grade US origin	InnovativeResearch	IGMSC57-K2 EDTA-Compl-10ml	Mouse plasma
Dulbecco's modified eagle medium (DMEM)	Sigma Aldrich	D6429-500ML
Dulbecco's phosphate buffered saline (DPBS)	Fisher Scientific	14190250	No calcium, no magnesium
Fetal bovine serum (FBS), research grade	Wisent	98150
LPS-EK	Invivogen	TLRL-EKLPS	Lipopolysaccharide from Escherichia coli K12
NIH/3T3 fibroblasts	ATCC	CRL-1658
Pam3CSK4	Invivogen	tlrl-pms	Synthetic triacylated lipopeptide – TLR1/2 ligand
Penicillin/streptomycin	Sigma Aldrich	P4333-100ML
Plasmocin	Invivogen	ANT-MPP	Mycoplasma elimination reagent
RAW-Blue cells	Invivogen	raw-sp	NF-κB/AP-1 reporter macrophage cell line
Trypan blue solution, 0.4%	Fisher Scientific	15250061
TrypLE express enzyme (1X)	Fisher Scientific	12604021	animal origin-free recombinant cell dissociation enzyme
Zeocin	Invivogen	ANT-ZN-1
Kits and assays
ELISA precoated plates, mouse IL-6	Biolegend	B213022
ELISA precoated plates, mouse TNF-α	Biolegend	B220233
Endotoxin (Escherichia coli) – Control standard endotoxin (CSE)	Associates of Cape Cope Inc.	E0005-5	Endotoxin for standard curve in chromogenic endotoxin assay
LAL water, 100 mL	Associates of Cape Cope Inc.	WP1001	Used with chromogenic endotoxin assay
Micro BCA protein assay	Fisher Scientific	PI23235
Limulus amebocyte lysate (LAL) Pyrochrome endotoxin test kit	Associates of Cape Cope Inc.	C1500-5	Chromogenic endotoxin assay reagent
QUANTI-Blue alkaline phosphatase detection medium	Invivogen	rep-qb2	Alkaline phosphatase assay to indirectly measure NF-κB/AP-1 activity
Polymeric coating reagents
Chloroform, anhydrous	Sigma Aldrich	288306-1L
Ethyl alcohol anhydrous	Commercial Alcohols	P006EAAN	Sigma: Reagent alcohol, anhydrous, 676829-1L
Straight tapered fine tip forceps	Fisher Scientific	16-100-113
Fluorinert FC-40 solvent	Sigma Aldrich	F9755-100ML	Fluorinated solvent for fPTFE
Cell culture grade water (endotoxin-free)	Fisher Scientific	SH30529LS
Poly(methyl methacrylate) (PMMA)	Sigma Aldrich	182230-25G
Sylgard 184 elastomer kit	Fisher Scientific	50822180
Teflon-AF (fPTFE)	Sigma Aldrich	469610-1G	Poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene]
Consumables
Adhesive plate seals	Fisher Scientific	AB-0580
Axygen microtubes, 1.5 mL	Fisher Scientific	14-222-155
Borosilicate glass scintillation vials, with white polypropylene caps	Fisher Scientific	03-337-4
Clear PS 48-well plate	Fisher Scientific	08-772-52
Clear TCPS 96-well plate	Fisher Scientific	08-772-2C
Clear TCPS 48-well plate	Fisher Scientific	08-772-1C
Cover glasses, circles	Fisher Scientific	12-545-81
Falcon tissue culture treated flasks, T25	Fisher Scientific	10-126-10
sticky-Slide 8 Well	Ibidi	80828
Superfrost microscope slides	Fisher Scientific	12-550-15
Tissue culture treated flasks, T150	Fisher Scientific	08-772-48