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Application Note

Measurement of IL-6 RNA levels in live cells using SmartFlare technology with the ImageStreamX Mark II Imaging Flow Cytometer
Introduction
Acute inflammatory responses to infection and tissue damage are orchestrated by release of cytokines, primarily from macrophages and mast cells, but also by the endothelium and tumor cells. A key initiator of the inflammatory response is tumor necrosis factor alpha (TNF-), which binds to its receptor on target cells and activates a pro-inflammatory transcriptional program via the transcription factor NF-B. A critical downstream target of NF-B is the gene encoding Interleukin-6 (IL6), which stimulates an array of anti-infection processes, including synthesis of acute phase proteins by the liver, proliferation of B cells, neutrophil production in bone marrow, and differentiation of Th17 helper T cells. Although beneficial in the context of attacking infection, dysregulation of IL-6 plays a deleterious role in autoimmune disease and cancer. In this study, we introduce a new method for measuring NF-B translocation by examining the increase in IL-6 mRNA levels in individual THP-1 (human acute monocytic leukemia) suspension cells using SmartFlare RNA detection probes in conjunction with imaging flow cytometry. The ability to detect RNA in live cells and to acquire multi-spectral images of large numbers of cells allows for the accurate assessment of IL-6 mRNA levels in THP-1 cells. Specifically, we used the ImageStreamX Mark II imaging flow cytometer to quantify the increase in IL-6 mRNA signal in TNF- and Interferon gamma (IFN-)stimulated and unstimulated THP-1 cells using IL-6 SmartFlare RNA detection probes. SmartFlare probes are internalized by live cells using existing endocytosis machinery, and, if the target mRNA is present, it hybridizes to the complementary capture strand on the SmartFlare probe, thereby releasing and unquenching a fluorescent marker. The probes can then exit the cells without adverse effects, allowing for subsequent downstream assays.

Data Sheet

Materials and Methods


THP-1 cells were seeded overnight at a density of 0.5 x 104 cells/mL in a 6-well plate with RPMI media with 5% FBS. The cell count was determined using the Scepter Cell Counter by EMD Millipore. IL-6 SmartFlare probe was added in the presence or absence of TNF- (20 ng/mL) and IFN-(10 ng/mL). All the chemicals were bought from EMD Millipore. The SmartFlare Uptake control, which has a fluorophore that is constitutively fluorescent, was used as a positive control probe, and the SmartFlare Scramble control, which targets nonsense mRNA sequences not present in

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cells, was used as the negative control. The SmartFlare Probes were added at a concentration of 100 pM. The degree of fluorescence was quantitated using the Bright Detail Intensity R7 (BDI) feature in the IDEAS data analysis software supplied with the ImageStreamX Mark II system. BDI sums the fluorescence of bright spots having a radius of 7 pixels or less within the cell imagery, thereby discriminating against uniformly distributed autofluorescence and nonspecific background signal.

Results
By measuring the intensity and number of bright spots in individual cells (BDI), we observed approximately 47% increase in BDI between TNF-/IFN- stimulated and unstimulated cells in IL-6 SmartFlare probe-treated samples, with punctate distribution of the IL-6 signal (Figure 1). The mean BDI increased from 2.77 x 104 to 4.08 x 104 and the MAD (Mean Absolute Deviation) changed from 1.39 x 104 to 2.39 x 104.

Low BDI-Unstim cells

Mean BDI-Stim cells

Normalized Frequency %

5 4 3 2 1 0

Unstimulated Stimulated

BF

SmartFlareCy5

BF

SmartFlareCy5

Mean BDI- Unstim cells

High BDI-Stim cells

1e4

2e4

3e4

Bright Detail Intensity R7

BF

SmartFlareCy5

BF

SmartFlareCy5

Figure 1. 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 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.

2.5 2 1.5 1 0.5 0

Unstimulated Stimulated

No significant increase in BDI was seen between the Unstimulated stimulated and unstimulated samples with Scramble 4 Stimulated controls, and the fluorescence intensity was much lower than that achieved using the IL-6 probe, demonstrating 3 the specificity of the IL-6 SmartFlare probe (Figure 2). Furthermore, fluorescence intensity of Uptake control 2 was consistent among all cells.

Conclusion
0 1e4 2e4 3e4 4e4 5e4 Bright Detail Intensity R7_M11_Ch11
X This 0 study demonstrates how the ImageStream Mark II 0 cytometer 1e5 2e5 with SmartFlare 3e5 imaging flow can be utilized Bright Detail Intensity R7_M11_Ch11 RNA detection probes to quantify upregulation of IL-6 mRNA by inflammatory stimuli in a monocyte-like cell line. The capability of SmartFlare probes to detect Uptake Control SmartFlare 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 the ImageStreamX Mark II imaging flow cytometer, the subcellular, punctate staining of the SmartFlare mRNA target can be discerned in addition to its abundance, even in very rare cells.

SmartFlare Scramble Control

SmartFlare-Cy5

Bright Detail Intensity


No stimulation TNF- + IFN- stimulation

SmartFlare-Cy5

Figure 2 (left).Ch01 Ch11 Ch11 Ch01 The specificity of IL-6 SmartFlare for the target of interest is evident in the difference in the fluorescence intensity between IL-6 and SmartFlare Scramble control probes. There is no significant change in Mean Bright Detail Intensity (BDI) between stimulated cells and unstimulated cells.

BF

BF

Mean Absolute Deviation


No stimulation TNF- + IFN- stimulation

SmartFlare IL-6 Probes SmartFlare Scramble Control

2.77E+04 4.23E+03

4.08E+04 5.35E+03

1.39E+04 1.35E+03

2.39E+04 1.02E+03

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.

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EMD Millipore, the M logo, Scepter, and SmartFlare are trademarks of Merck KGaA, Darmstadt, Germany. Imagestream is a registered trademark of Amnis Corporation. All trademarks belonging to third parties are the properties of their respective owners. Lit No. AN5774EN00 BS-GEN-13-08317 Printed in the USA. 05/2013 2013 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.