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.
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.
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 responses and produce antimicrobial proteins and compounds, cytokines and chemokines, and present antigens2,3. Alternatively activated macrophages play a role in regulating immunity, maintaining tolerance, tissue repair, and wound healing4-8. Because of their wide array of functions, macrophages can play a role in numerous diseases including atherosclerosis, arthritis, and cancer9. Thus, the study of macrophages has been a key area of research for some time in a wide variety of disease fields.
Despite their importance in the innate immune response, macrophages can be challenging cells to work with. In particular, it is difficult to obtain efficient transfection using lipid reagents in macrophages without associated toxicity10,11. Moreover, even when siRNA is efficiently delivered to macrophages, the robustness of RNAi-induced gene knockdown often can be fairly moderate and can vary from gene to gene.
To overcome these technical challenges, we have optimized transfection and knockdown techniques12-16 in two mouse macrophage cell lines, RAW264.717 and J774A.118. This approach uses the Amaxa Nucleofector 96-well Shuttle System for transfection; this system uses a combination of specialized reagents and electroporation to transfect cells in 96-well format19. Following transfection, we describe methods to maximize cell recovery and viability and to maximize subsequent siRNA-induced gene knockdown. To illustrate the utility of this approach, we describe a protocol for siRNA delivery to these macrophage cell lines, stimulate these cells with lipopolysaccharide (LPS), and monitor the innate immune response at the level of production of several pro-inflammatory cytokines. We provide sample data in which we target the Toll-like receptor (TLR) family, whose members sense LPS and other pathogen associated molecular patterns (PAMPs), to regulate innate immunity.
1. Maintenance of Macrophage Cell Lines
2. Programming the 96-well Shuttle System for Transfection
3. Preparing the Reagents for Transfection
4. Preparing the Macrophages for Transfection
5. Nucleofection of Macrophages with siRNA
6. Recovery and Plating of Macrophages
7. Stimulation of Macrophages with LPS
8. Monitoring the Induced Innate Immune Response, Gene Knockdown, and Viability in Macrophages
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 different PAMPs, with LPS from Gram negative bacteria recognized by TLR420, lipopeptides such as PAM3CSK4 recognized by TLR221, and dsRNA from viruses such as the mimic poly(I:C) recognized by TLR322,23. As shown in Figure 2, inhibition of TLR4 (but not other TLRs using siRNA) strongly diminishes production of LPS-induced IL-6 and TNFα. Moreover, inhibition of IL-6 with siRNA also diminishes IL-6 production while not affecting TNFα production (compare panel A için B videodan Figure 2).
Figure 1: FITC-labeled siRNA is efficiently transfected into the mouse macrophage cell line J774A.1 using the described methods. Immediately after transfection, the cells were washed several times, fixed in paraformaldehyde, and fluorescence was monitored by flow cytometry. The figure compares cells incubated in the presence of FITC-siRNA that were either transfected (blue curve) or not (red curve).
Figure 2: Efficacy and specificity of siRNA in the mouse macrophage cell line RAW264.7. RAW264.7 cells were transfected with the indicated siRNAs (either control non-targetting siRNA pools #1 or 2 or siRNAs that target TLR4, TLR3, TLR2, or IL-6 as indicated). 24 hr later, cells were stimulated with 20 ng/ml LPS for 6 hr and cytokine production (either IL-6 in panel A or TNFα in panel B was subsequently monitored by ELISA. Asterisks indicate value significantly different from control treatment (p <0.05, t-test).
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, which exhibits more consistency in gene knockdown and limited effects on viability (this more robust system is more expensive than lipid-mediated approaches). Many variables were tested in order to optimize these procedures. We were able to use this system to efficiently deliver siRNA to many different primary macrophages and immortalized macrophage cell lines (in some cases this necessitated using the Amaxa 96-well cell line optimization kit). In preliminary tests with these different mouse macrophage cell lines (RAW264.7, J774A.1, and MH-S) and primary cells (thioglycollate-elicited and bone marrow-derived macrophages), we found that RAW264.7 and J774A.1 cells exhibited the most robust siRNA-induced gene knockdown (followed by MH-S and primary cells, respectively, data not shown). As these two immortalized macrophage cell lines are commonly used for study, we optimized our RNAi procedures further using these lines.
To monitor transfection efficiency, we either use the control GFP plasmid in the nucleofector kits or FITC-labeled siRNA. The GFP plasmid exhibits strong fluorescence the day after transfection, with transfection efficiency of 30-50% as assayed microscopically or by flow cytometry (data not shown). With FITC-labeled siRNA, we observe >99% transfection efficiency as assayed by flow cytometry (dsRNA transfects better than plasmid DNA). While it is important to wait for fluorescence to develop when using the GFP plasmid, the fluorescent siRNA should be monitored relatively soon after transfection because the fluorescent signal weakens with time.
Transfection under the described conditions does not have a significant impact on cell viability. We typically monitor cell viability following our siRNA treatments using fluorescein diacetate. This compound is not fluorescent; however, when cleaved by cellular esterases in live cells, it becomes fluorescent. Moreover, the fluorescent form is then retained in cells with an intact plasma membrane24. This fluorescence-based assay is a relatively straightforward, rapid, and cheap assay to monitor cell viability, although there are other more sophisticated assays that for example monitor cellular ATP levels.
We typically assay our cells for innate immune function 24-36 hr following transfection, which is on the early side for RNAi compared to other cell lines. While one has to wait for endogenous proteins to turn over to facilitate gene knockdown, one must balance this with loss of siRNA potency, and we find more robust knockdown in these macrophage lines at slightly early times than the typical 48-72 hr used in other cell types. While it is preferable to monitor protein levels by western blot, we typically monitor mRNA levels by qPCR, with the goal of studying multiple genes at once, for which multiple antibodies and western blots may not be practical. We use LPS at the minimal dose that maximizes inflammatory cytokine production.
When targeting new genes with siRNA, we start with the relatively high siRNA dose described in the Protocol and then titrate down siRNAs that induce a phenotype. Subsequent controls designed to avoid off-target effects include the use of multiple siRNA duplexes to determine if more than one siRNA can induce the same phenotype, verifying gene knockdown by qPCR or western blot, and using gene overexpression studies to complement the RNAi approach. These approaches should allow for rapid identification of genes that regulate innate immunity in mouse macrophages.
The authors have nothing to disclose.
Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.
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 electrooration | Invitrogen | 13750062 | |
Ultrapure E. coli O111:B4 LPS | List Biological Laboratories | 421 | |
DMEM, high glucose | Invitrogen | 11995-065 | |
RPM1-1640 | 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 TNFa Duoset ELISA kit | R&D Biosystems | DY410 | |
Fluorescein diacetate | Sigma-Aldrich | F7378 | |
RLT Bufer | Qiagen | 79216 | |
Rneasy mini kit | Qiagen | 74134 | |
Vybrant Phagocytosis Assay Kit | Invitrogen | V-6694 |