Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.
Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.
Transfection of nucleic acids in cells has a diverse application in scientific research. Examples include (1) reporter genes to study the role of different gene elements in gene expression, (2) protein expression plasmids to overexpress the protein of interest, and (3) small interfering RNA to downregulate gene expression. By manipulating the expression level of particular genes and measuring the differential effect of such manipulations, researchers can deduce the gene functions in the chosen biological systems. Not all transfection methods provide the same transfection efficiencies, and even the same transfection method does not transfect all cell types equally1. Hence, different transfection methods have been developed such as calcium phosphate method, DEAE dextran, cationic lipid transfection, cationic non-lipid polymer transfection, electroporation, and nucleofection2,3.
Transfection into macrophages is especially difficult due to the fact that macrophages are professional phagocytes that are very sensitive to foreign materials including bacteria derived (methylated) DNA4. Introduction of foreign DNA activates the Toll-like receptor 9 (TLR9) pathway leading to the production of cytokines and nitric oxide5,6. These activated macrophages may then be less responsive to treatment that the researchers intend to examine.
Our lab routinely transfects the RAW264.7 macrophage cell line with luciferase reporter genes, and we have developed a protocol that is robust enough to have luciferase signal significantly higher than background, but also gentle enough for macrophages to remain at their resting state. The behaviours of the transfected cells were evaluated by a firefly-luciferase reporter gene harbouring the promoter region of IκBζ (pGL3- IκBζ). IκBζ expression is upregulated by the bacterial cell wall component lipopolyssacharide (LPS)7,8, and downregulated by the anti-inflammatory cytokine, Interleukin-10 (IL-10)8. To account for transfection variation among wells, we co-transfect a control plasmid containing the Renilla luciferase gene (e.g., phRL-TK) for normalization purposes. The protocol described is optimized after testing various parameters including timing of transfection, type of transfection reagents, amounts of transfection reagents and of plasmid DNA, as well as ratio of transfection reagent to plasmid DNA. The two transfection reagents included in this protocol are (1) a lipid-based transfection reagent and (2) a protein/polyamine-based transfection reagent.
1. Plasmid DNA Purification
2. Cell Culturing and Seeding
3. Transfection
4. Cell Stimulation and Luciferase Assay
5. Data Analysis
Figure 1 compares the transfection efficiency of the two transfection reagents in RAW264.7. The lipid-based reagent typically gave about 25% transfection rate while the protein/polyamine-based transfection resulted in about 5% efficiency (Figure 1A). The difference in transfection efficiency was also observed in luciferase signals in RAW264.7 cells transfected with the pGL3-IκBζ promoter reporter (Figure 1B). Addition of LPS to these transfected cells increased firefly luciferase signal, a direct indication of increased transcription activity of the IκBζ promoter reporter. In other words, the results suggested that LPS upregulated the expression of the IκBζ gene, consistent with previous reports7,8. Figure 2 shows the typical results obtained in our experiments, using lipid-based transfection as an example. After obtaining individual signal values from firefly luciferase and Renilla luciferase (Figures 2A and 2B), the firefly luciferase signals were normalized to the Renilla luciferase signal (Figure 2C). Normalization is recommended due to well-to-well variation in transfection efficiency. To determine if the treatment conditions (LPS or LPS+IL-10) altered the reporter signals, the firefly:Renilla ratio (i.e., the normalized signals) of the treatment groups were compared with that of the untreated (unstimulated) sample (Figure 2D). Our data showed that treating RAW264.7 cells with LPS upregulated the activity of the pGL3-IκBζ promoter reporter, indicating that LPS increased the transcription level of the IκBζ gene. In the presence of IL-10, the IκBζ promoter reporter had lower activity, suggesting that IL-10 was able to inhibit LPS-induced transcription of the IκBζ gene. The protein/polyamine-based transfection usually gave lower values in both firefly luciferase and Renilla luciferase signals. Figure 3 compares the length of rest time between transfection and stimulation (24 hr or 48 hr), and shows that luciferase signals decreased over time. The decrease in signal did not interfere with data interpretation when the lipid-based transfection reagent was used (Figure 3A); induction by LPS and inhibition by IL-10 were still observed after 48 hr of rest. However, the decrease in signal was more significant when the protein/polyamine-based transfection reagent was used (Figure 3B), especially the Renilla luciferase signals. As a result, the difference between treatment groups was not observed after a 48 hr rest.
One undesired effect of transfection is cell death so the impact of each transfection method on the degree of apoptosis was assessed. Cells were transfected, or not, and subjected to Annexin-V and propidium iodide (PI) staining. Lysates prepared from these cells were also analyzed for the presence of intact and cleaved poly ADP ribose polymerase (PARP) protein. Flow cytometric analysis of the Annexin-V/PI stained cells suggested that the lipid-based transfection slightly increased the proportion of Annexin-V positive cells as compared to the untransfected cells, while the protein/polyamine-based transfection did not (Figure 4A). Similarly, the lipid-based transfected slightly increased the proportion of cleaved PARP while the protein/polyamine-based transfection did not (Figure 4B). However, despite the elevated numbers of apoptotic cells, the morphology of the cells by light microscopy did not change (Figure 4C). More importantly, the biological response of the lipid-based transfected cells remained identical to those of the untransfected cells (Figure 4D). The cells were stimulated with LPS ± IL-10, and the amounts of TNFα secreted into the culture supernatant were quantified by ELISA. Both the untransfected and lipid-based transfected cells made similar amounts of TNFα in response to LPS and were inhibited by the addition of IL10. No TNFα was made when the cells were not stimulated (Figure 4D) suggesting that the cells remained naïve after transfection.
Figure 1. The lipid-based transfection gave higher transfection efficiency than the protein/polyamine-based transfection. (A) RAW264.7 cells were transfected a GFP-expressing plasmid with either the lipid-based or the protein/polyamine-based transfection reagents. GFP signal was measured by flow cytometry after 24 hr. (B) RAW264.7 cells were transfected with the pGL3-IkBζ promoter reporter. After 48 hr rest, cells were stimulated with LPS for 6 hr. Firefly luciferase signal was measured according to manufacturer’s instructions. Please click here to view a larger version of this figure.
Figure 2. Typical luciferase assay data. RAW264.7 cells were transfected with TK-Renilla and IkBζ promoter reporter. After 24 hr rest, cells were stimulated with LPS ± IL-10 for 2 hr. (A) Firefly luciferase and (B) Renilla luciferase signals were measured according to luciferase assay manufacturer’s instructions. (C) Reporter activity was normalized to the TK-Renilla signal and plotted as Firefly/Renilla ratio. (D) Fold change is calculated by dividing the firefly:Renilla ratio of the stimulated samples by that of the unstimulated sample. Please click here to view a larger version of this figure.
Figure 3. The strength of the luciferase signal is time-dependent. (A) Lipid-based transfected and (B) Protein/polyamine-based transfected cells were rested for either 24 hr or 48 hr prior to stimulation with LPS ± IL-10 for 2 hr. Please click here to view a larger version of this figure.
Figure 4. Effect of transfection on RAW264.7 cells. (A) RAW264.7 cells were transfected with the IkBζ promoter reporter. After 24 hr rest, cell death was assessed by Annexin-V and propidium iodide (PI) staining on a flow cytometer. (B)Transfected cells were subjected to immunoblotting analysis for PARP (full length and cleaved) and GAPDH (loading control). Band intensities were then quantified using imaging software. L = lipid-based transfection, P = protein/polyamine-based transfection, STP = 0.1 µM staurosporin. (C) Microscope images of cells transfected with the lipid-based transfection reagent. (D) Cells transfected with the lipid-based transfection reagent were stimulated with LPS ± IL-10 for 6 hr, and the levels of the pro-inflammatory cytokine TNFα were measured using ELISA. Please click here to view a larger version of this figure.
The protocol described here does not solely focus on transfection efficiency, but aims to strike a balance between efficiency and preservation of the physiological states of the cells. Specifically, our procedure succeeds in minimizing the toxicity of transfection reagent and maximizing luciferase signal.
One critical step in the protocol is the health of the cells. Overgrown cultures are not suitable for transfection as their physiology changes, and continuous culturing of RAW264.7 cells for a long period of time can also change the phenotype and function of the cells10. Freshly thawed cells that have a low passage number are recommended to use for transfection.
Another important consideration is the choice of transfection reagents. Lipid-based transfection reagents are typically used in research due to its ease of use and commercial availability. However, some of these reagents caused unintended (and usually unwanted) changes in global gene expression in transfected cells11,12. To address this problem, cells are only incubated with the transfection reagents for a few hours instead of the typical O/N, or a non-lipid-based reagent is chosen for transfection. Longer incubation time with the transfection reagent will increase transfection efficiency, but it can also be harmful to the cells either causing cell death or cell activation, both of which can interfere with the experimental design. Figures 4A and 4B show that the protein/polyamine-based transfection did not cause apoptosis or necrosis, compared to untransfected cells. The lipid-based transfection, even with the short incubation time, caused higher level of cell death. It is correlated to the higher transfection efficiency of the lipid-based transfection reagent. However, when the transfected cells were observed under the microscope, there were no notable morphological changes (Figure 4C). Activated macrophages usually adopt a sprawling shape, but both the untransfected and transfected cells did not show signs of activation prior to stimulation, indicating that they were in their resting states. In addition, the transfected cells responded similarly to LPS and IL-10 stimulation as the untransfected cells (Figure 4D). These observations collectively indicate that this transfection procedure does not alter the cells’ natural behaviour.
The time between transfection and experimental treatment (the rest time) is also crucial. Enough time needs to be given for the luciferase genes to express and for the cells to re-establish their resting state; however, a long incubation can reduce luciferase signals, especially when transfection efficiency is not high.
The procedure here applies to the specific luciferase reporter gene used in our lab. Minor adjustments will be needed when another reporter is used. Parameters that need to be tested include different firefly luciferase reporter:Renilla luciferase ratio, the rest time between transfection and treatment, and treatment conditions. A 50:1 ratio of pGL3-IκBζ: phRL-TK is typically used, but the ratio can range from 1:1 to 100:1. It was found that a 24 hr rest between the removal of transfection solution from the cells and the start of experimental treatment gave the best signal. After 48 hr, luciferase signals started declining, and differences between treatment groups were reduced or even abolished.
The described transfection procedure is not limited to luciferase reporter assays. Other experimental designs that do not need a homogenous population will be benefited; for instance, fluorescent microscopy which relies on observations from individual cells. One disadvantage will be to locate the successfully transfected cells among untransfected counterparts, but the benefits of being less time-consuming and labor-intensive may be more desired. In cases where stable cell lines are preferred, our protocol provides a mean for initial experiments in which experimental procedure can be optimized when stable cell lines are being generated.
The authors have nothing to disclose.
This study was funded by Canadian Institutes for Health Research (CIHR) grant. STC holds a doctoral research award from the CIHR and the Michael Smith Foundation. EYS holds a CIHR scholarship. The CIHR Transplantation Training Program also provided graduate scholarships to STC, EYS and SS.
PureLink HiPure Plasmid Maxiprep Kit | Life Technologies | K210007 | Any maxiprep kit will work |
Phenol:chloroform:isoamyl alcohol | Life Technologies | 15593-049 | Molecular Biology Grade. Phenol is toxic so work in the fume hood, if possible. Use the lower clear organic layer if two layers of liquid form in the container. |
DMEM | Thermo Scientific | SH30243.01 | Warm in 37°C water bath before use. |
Fetal Bovine Serum | Thermo Scientific | SH30396.03 | Inactivated at 56°C water bath for 45 minutes before use. |
Opti-MEM | Life Technologies | 31985-070 | Warm to at least room temperature before use. |
XtremeGene HP DBA transfection reagent | Roche | 6366236001 | Warm to room temperature before use. |
GeneJuice | EMD Millipore | 70967 | Warm to room temperature before use. |
5X Passive Lysis Buffer | Promega | E1941 | 30 ml is included in the Dual Luciferase Reporter Assay System |
Dual Luciferase Reporter Assay System | Promega | E1910 |