Infections and tissue damage lead to acute inflammatory responses, orchestrated by the release of cytokines from macrophages and mast cells, as well as endothelium and tumor cells. Tumor necrosis factor alpha (TNF-α) is one cytokine that plays a critical role in initiating an inflammatory response, by binding to its receptor on target cells and activating a pro-inflammatory transcriptional program via the transcription factor NF-κB.
A key downstream target of NF-κB is the gene encoding interleukin-6 (IL-6). IL-6 stimulates various anti-infection processes, including the differentiation of Th17 helper T cells, proliferation of B cells, neutrophil production in bone marrow, and the liver’s synthesis of acute phase proteins. However, though beneficial in the context of attacking infection, problems with IL-6 regulation can lead to autoimmune disease and cancer.
A new technique is available for measuring NF-κB translocation by examining the increase in IL-6 mRNA levels in individual human acute monocytic leukemia (THP-1) suspension cells. The approach combines SmartFlare™ RNA detection probes (EMD Millipore, Billerica, MA) with imaging flow cytometry. Specifically, the ImageStream®X Mark II imaging flow cytometer (EMD Millipore) was used to quantify the increase in IL-6 mRNA signal in TNF-α and interferon-gamma (IFN-γ)-stimulated and unstimulated THP-1 cells using IL-6 SmartFlare RNA detection probes. The probes’ ability to detect RNA in live cells, combined with the capacity to acquire multispectral images of large numbers of cells, allowed for the accurate assessment of IL-6 mRNA levels in THP-1 cells.
Materials and methods
THP-1 cells were seeded overnight at a density of 0.5 × 106 cells/mL in a six-well plate with RPMI media with 5% fetal bovine serum (FBS).
SmartFlare RNA detection probes
IL-6 SmartFlare probe was added in the presence or absence of TNF-α (20 ng/mL) and IFN-γ (10 ng/mL). The positive control probe used was the SmartFlare uptake control, which has a constitutively fluorescent fluorophore. The negative control was the SmartFlare scramble control, which targets nonsense mRNA sequences not present in cells. The probes were all added at a concentration of 100 pM.
A SmartFlare probe is a reagent capable of detecting specific mRNAs and miRNAs in live, intact cells. Figure 1 shows the probe’s structure and mechanism of action. Each one is made of a gold nanoparticle conjugated to many copies of the same double-stranded oligonucleotide encoding the target sequence. One strand of the oligonucleotide bears a fluorophore that is quenched by its proximity to the gold core. When the nanoparticle comes into contact with its target, the target RNA displaces the fluorophore. The reporter strand, now unquenched, fluoresces and can be detected using any fluorescence detection platform.
Figure 1 – Molecular mechanism of SmartFlare RNA detection probe.The probes are internalized by live cells using existing endocytosis machinery. They require no upfront sample preparation, and have no toxic effects on cellular fate, as well as no known nonspecific, off-target effects. Once the detection and relative quantitative analysis of RNA levels are complete, the probes can then exit the cells without adverse effects, allowing for subsequent downstream assays. This allows researchers to assess multiple biomarkers or downstream functionalities in the same cells.
Compared to currently used methods for studying RNA that involve examination of non-native, amplified RNA targets, SmartFlare probes have the potential to provide results that show greater correlation to in vivo observations. In vivo, most cells reside in heterogeneous tissues, and cell-to-cell variation in gene expression can be subtle yet crucial for tissue function. SmartFlare technology can be used to quantitate RNAs in individual cells, providing previously unobtainable gene expression information that distinguishes one heterogeneous cell population from another with high resolution.
Finally, a typical SmartFlare probe exhibits specificity for its target, as evidenced by the increase in fluorescence upon addition of the target sequence, and lack of signal when a nontarget sequence is added in equal amounts.
Fluorescence detection
The degree of fluorescence was quantitated with the ImageStreamX Mark II system, using the Bright Detail Intensity R7 (BDI) feature in its IDEAS data analysis software. BDI sums the fluorescence of bright spots having a radius of 7 pixels or less within the cell imagery. This discriminates against uniformly distributed autofluorescence and nonspecific background signal.
Results
The intensity and number of bright spots in individual cells (BDI) was measured, and an approximately 47% increase in BDI between TNF-α/IFN-γ stimulated and unstimulated cells in IL-6 SmartFlare probe-treated samples, with punctate distribution of the IL-6 signal, was observed (Figure 2). The mean BDI increased from 2.77 × 104 to 4.08 × 104 and the MAD (mean absolute deviation) changed from 1.39 × 104 to 2.39 × 104 (Table 1).
Figure 2 – Visualization and quantification of IL-6 mRNA in THP-1 cells shows increased IL-6 mRNA levels in cytokine-stimulated cells. Mean bright detail intensity (BDI) showed an approximately 47% increase in stimulated cells compared to unstimulated cells. The increased IL-6 level is also confirmed by the clear shift in the overlaid histograms showing BDI of stimulated and unstimulated cell populations.Table 1 – Summary of quantification of fluorescent signal intensity in stimulated and unstimulated THP-1 cells treated with IL-6-specific and scramble control SmartFlare probes
No significant increase in BDI was seen between the stimulated and unstimulated samples with scramble controls. In addition, the fluorescence intensity was much lower than that achieved using the IL-6 probe, demonstrating the specificity of the IL-6 SmartFlare probe (Figure 3). Finally, fluorescence intensity of uptake control was consistent among all cells.
Figure 3 – The specificity of IL-6 SmartFlare probe for the target of interest is evident in the difference in fluorescence intensity between IL-6 and SmartFlare scramble control probes. There is no significant change in BDI between stimulated cells and unstimulated cells.Conclusion
This study demonstrated how the ImageStreamX Mark II imaging flow cytometer can be utilized with SmartFlare RNA detection probes to quantify upregulation of IL-6 mRNA by inflammatory stimuli in a monocyte-like cell line. The capability of the probes to detect changes in specific RNAs in live cells allows researchers to capture gene regulation events at the single cell level by flow cytometry. When used with an imaging flow cytometer, the subcellular, punctate staining of the mRNA target can be discerned in addition to its abundance, even in very rare cells.
The probes have a variety of other potential uses as well, including the sorting of cells based on gene expression (enabling even higher levels of enrichment using additional intracellular markers), live cell tracking of RNAs, and detection of multiple types of biomolecules (such as protein + RNA) in the same sample. The technology can also be used for multiplexed detection of up to three different RNAs (using different fluorophores), enabling the normalization of gene expression levels to “housekeeping” or “control” genes within individual cells. In summary, by enabling the researcher to discover cells that express particular genes at particular levels in real time, the probes can truly enhance the value of data obtained for RNA analysis.
Shobana Vaidyanathan is a Research Scientist, Amnis Corp., A Part of EMD Millipore, 645 Elliot Ave. West, Ste. 100, Seattle, WA 98119, U.S.A.; tel.: 206-576-7151; e-mail: [email protected].