Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

The protocol outlined here presents a reliable and efficient way to transfect THP-1 macrophages which are usually rather difficult to transfect. Transfection can be achieved with transfection efficiencies of above 90% for siRNA without significant reduction of cell vitality. Efficiencies for plasmids may be less due to their size but transfection efficiencies of around 70% can be usually achieved. The efficiency of siRNA-mediated knockdown can reach 80 to 90% depending on the applied siRNA. A major advantage of this protocol is that it does not interfere with cell differentiation. After transfection the cells still respond normally to the differentiation agent PMA (Figure 1A a 1B and Figure 4)¹². In addition cellular polarization by cytokine stimuli such as IL10 or LPS/IFNγ is unaffected¹², although this also depends strongly on the medium selected for cultivation after transfection as shown in Figure 4.

There are several options within the procedure which can be modified in order to adapt the protocol to specific requirements. As shown in Figure 4 the cells react differently to the same IL10 stimulus depending on the culture medium selected after transfection. This indicates that the medium composition has strong influence on cellular behavior. As the effect imposed by the medium might differ for various experimental stimuli, it is possible that the optimal medium might change and therefore there is a need to verify the suitability of the medium for different experiments independently. However the RPMI-1640 medium, which is the default medium used for cultivation of THP-1 cells, has proven to be ill-suited for cultivation after transfection as a significant loss in cell vitality was observed. Furthermore the protocol can also be applied to THP-1 monocytes without prior PMA-induced differentiation, this obviously removes the need for cell detachment during the procedure but all remaining steps do not have to be adjusted. The THP-1 monocytes can be differentiated afterwards if required.

The most critical elements of the protocol are firstly the detachment and secondly the time required for transfection. The detachment must be performed as gently as possible, in order to maintain high cell viability. Therefore we recommend the comparatively mild enzymatic detachment by Accutase I over quite aggressive methods such as trypsinization. Further detachment methods such as treatment with Lidocain or scraping were found to be rather detrimental and should not be used. In the case that 30 min of Accutase I treatment should not be sufficient to detach all cells properly this is usually an indication of incorrectly stored or expired Accutase I solution or of too many freeze-thaw cycles. We have obtained good results by storing small aliquots of Accutase I at -20 °C to reduce the number of freeze-thaw cycles. However when there is insufficient detachment either replace Accutase I with fresh Accutase or increase incubation time up to 1 hr. In addition gentle tapping or rinsing of the flask or plate can assist detachment. Regarding the required time for transfection the exposure time of the cells to pure Nucleofector solution has got high impact on cell survival and thus should be as short as possible. The best way to achieve this is to perform each transfection at a time (steps 2.10 to 2.15) and not multiple transfections in parallel.

If knockdown efficiency is low, this usually is not caused by low transfection efficiency, but this can be easily verified using flow cytometry or fluorescence microscopy and fluorescently labeled siRNA or a GFP encoding plasmid. Mostly the cause is an inefficient siRNA or plasmid DNA. Under these circumstances it should be considered to increase the amount of siRNA (up to 2-3 µg) or plasmid DNA (up to 1-2 µg) or to use a different siRNA or expression vector if available. Alternatively, satisfactory results might also be achieved by using a pool of several different siRNAs directed against the same target. Furthermore, time course experiments might be required to accurately determine the period of maximal effect, which is usually reached after 24 to 72 hr after transfection.

Apart from transfection by electroporation there are further well established techniques for transfection of mammalian cells. Frequently applied systems are chemical transfection agents which can form complexes with the cargo nucleic acid and then facilitate the transport into the cells. The most commonly used reagents are either based on different lipid species or can be chosen from a number of cationic polymers³. For both approaches a diverse selection is commercially available. These transfection reagents offer the advantage that they are usually easy to use, do not require much time, and work with adherent cells as well, thus removing the necessity for detachment. Unfortunately, macrophages are rather difficult to transfect by these methods as they do not proliferate significantly in vitro and possess defense mechanisms directed against foreign cytosolic DNA^13-15. Thus, chemical transfection approaches frequently result in a severe reduction of cell viability. However as shown in Figure 2 there are chemical transfection agents which can successfully transfer siRNA into macrophages, but as flow cytometric (Figure 2A) and fluorescent microscopic (Figure 2B) analyses indicate they fail to achieve the same homogenous distribution of siRNA within the cells as obtained by Nucleofection. In fact, the flow cytometric data indicate that two different populations of transfected cells occur, firstly a population with similar fluorescence as the cells after Nucleofection is detected and secondly there is a population with more intense fluorescence. Definite confirmation is still required but we suppose that the first population is in the fluorescence microscopy identical with those cells which show a homogenous distribution of fluorescent siRNA within the cytosol while the second population likely corresponds to the cells with very bright agglomerates. The flow cytometric data shown in Figure 2A suggest that nucleofection results in less siRNA per cell than the alternative lipofection approach. However the data in Figure 3 shows that even the comparatively small amount of siRNA incorporated by Nucleofection is already sufficient for 80 to 90% knockdown of the target gene. Therefore the additional amount of siRNA incorporated by the second population after chemical transfection is very likely surplus and is thus more likely to cause undesired side effects than to further increase knockdown efficiency. Apart from that formation of intracellular agglomerates is already undesirable in itself since those siRNA molecules are likely not functional or at least less efficient than free siRNA molecules and might cause off target effects. Furthermore the true nature of these agglomerates is not yet clear. It is possible that these bright spots represent endosomes indicating that the siRNA was internalized by the cells but subsequently release from the endosomes failed and the siRNA remains trapped and therefore ineffective. Alternatively the spots could be agglomerates which are formed intracellularly. Functional evaluations of the chemical transfection are pending, therefore no definitive statements on knockdown efficiency can be made yet, still the existence of two distinct populations is already unfavorable as this means that all results describe the mean of both populations, this does therefore not correspond to either of the populations. For that reason Nucleofection is the superior approach.

Nevertheless there are also limitations to the transfection procedure outlined here. Since the cells have to be in suspension for Nucleofection adherent cells need to be detached, which presents an additional stress factor to the cells. Furthermore the entire protocol is rather time consuming. Since transfections cannot be performed simultaneously, but have to be performed quickly in order to avoid cell damage, the number of samples is limited to about 8 – 10 per experiment. Thus, this protocol is not suited for high throughput screening projects. For high throughput applications different Nucleofector systems are available. Firstly, there is the 4D-Nucleofector system that offers a 16 well strip format. For even larger throughput, there are the 96 well Shuttle system and the 384 well HT Nucleofector system. However as these newer systems work with conductive polymer electrodes instead of aluminum and therefore electrical conditions, i.e. programs and composition of Nucleofector solution, have changed accordingly, it needs to be determined if our protocol can be transferred to those systems.

However, despite these limitations the protocol presented here does yield a reliable and effective transfection of THP-1 cells which is superior to all other non-viral approaches. This protocol enables the investigation of the effects of altered gene expression in THP-1 cells which in all other aspects differentiate and polarize normally and thus show as little side effects of transfection as possible.