Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Summary

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Abstract

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

Introduction

In this protocol, we describe efficient methods to inhibit genes in mouse macrophage cell lines using siRNAs and monitor the effects of these treatments on the innate immune response. These procedures are performed in 96-well format, allowing for RNAi screens in medium- or high-throughput fashion.

In response to infection, humans mount an immediate innate immune response and a slower but more specific adaptive immune response. This rapid innate immune response involves the recruitment and activation of phagocytic innate immune cells including macrophages¹. Classically activated macrophages are involved in acute inflammatory respo....

Protocol

1. Maintenance of Macrophage Cell Lines

1. Grow RAW264.7 and J774A.1 cell lines in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 ºC in the presence of 5% CO₂. To help maintain sterility, add 1% penicillin/streptomycin (pen/strep) to the media (although this is not strictly necessary).
2. Critical step: Ensure that the macrophages are growing in a healthy state for efficient transfection and siRNA-induced gene knockdown. Use cells between passages 3 and 10 after fresh thawing, as these macrophage lines exhibit the best transfection efficiency and maintain robust post-transfection survival at this stage; do no....

Results

To demonstrate the efficiency of transfection using this approach, we monitored uptake of FITC-labeled siRNA using a flow cytometer (Figure 1).

To illustrate the utility of our approach for monitoring the innate immune response, we transfected siRNAs targeting known innate immune regulatory genes into the RAW264.7 macrophage cell line, stimulated the cells with LPS, and then monitored production of the pro-inflammatory cytokines IL-6 and TNFα. Different TLRs recognize differen.......

Discussion

Numerous studies have been published in which individual genes have been targeted by siRNA in murine macrophages. While lipid-mediated transfection has been used to deliver siRNA to macrophage cell lines on an individual basis, these methods suffer from potential effects on viability, limited gene knockdown, and variability from gene to gene. To develop more robust assays that could be used to target genes in medium- or high-throughput fashion, we optimized techniques using the Amaxa nucleofector 96-well shuttle system, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amaxa nucleofector 96-well shuttle system	Lonza	AAM-1001S
Amaxa SF cell line 96-well nucleofector kit	Lonza	V4SC-2096
RAW264.7 mouse macrophage cell line	ATCC	TIB-71
J774A.1 mouse macrophage cell line	ATCC	TIB-67
siGenome smartpool siRNA	Dharmacon	varies depending on gene
Non-targeting control siRNA pool	Dharmacon	D-001206-13-20
Block-iT fluorescent oligo for electroporation	Invitrogen	13750062
Ultrapure E. coli O111:B4 LPS	List Biological Laboratories	421
DMEM, high glucose	Invitrogen	11995-065
RPMI-1640	Invitrogen	11875-093
Penicillin-streptomycin solution (Pen/Strep)	Fisher	SV30010
0.25% Trypsin-EDTA	Invitrogen	25200072
96-well tissue culture plates	Fisher	07-200-89
96-well round bottom sterile plates (not coated)	Fisher	07-200-745
Mouse IL-6 Duoset ELISA kit	R&D Biosystems	DY406
Mouse TNFα Duoset ELISA kit	R&D Biosystems	DY410
Fluorescein diacetate	Sigma-Aldrich	F7378
RLT buffer	Qiagen	79216
RNeasy mini kit	Qiagen	74134
Vybrant Phagocytosis Assay Kit	Invitrogen	V-6694