Quantitation of Nuclear Translocation Events using ImageStream Multispectral Imaging Cytometry

Thaddeus George, Brian Hall, Cathy Zimmerman, Keith Frost, Mike Seo, Dave Perry, William Ortyn, David Basiji and Philip Morrissey

Background and Summary

The nuclear factor -kB (NF-B) proteins comprise a small group of closely related transcription factors. While they were originally characterized as DNAbinding proteins with specificity for the immunoglobulin enhancer and enhancing gene transcription, subsequent studies showed the NF-kB proteins to function as part of a transcription activation system affecting a different genes in multiple tissues. have emerged as prime targets for both basic research and drug discovery. Drug discovery efforts to date have focused on a variety of components or processes in the NF-B system. Examples include inhibiting DNA binding by NF-B, preventing degradation of the inhibitory IB complex and directly inhibiting IKK kinase activity. For the analysis of NF-B activation and translocation, the ImageStream system offers a uniquely powerful solution. In this report, we show how the use of the two central capabilities of the ImageStream system -- high resolution imaging and flow-based population analysis -- can produce a robust quantitative analysis of translocation of NF-B from the cytoplasm to the nucleus in cells stimulated in by two different protocols.

Quantitation of Nuclear Translocation Events

Members of the NF-B family were linked, shortly after their discovery, to chronic and acute inflammatory diseases. More recently, evidence has indicated that aberrant regulation of this family could also be associated with autoimmune diseases and different types of cancer. It is not surprising then, given the number of major ailments with which NF-B has become associated, that these proteins

Results
Visualization of NFkB Nuclear Translocation in A549 Cells Using Immunofluorescence Microscopy TNF-a and IL-1b stimulation initiates a signaling cascade that results in the translocation of NF-B from the cytoplasm to the nucleus of the adherent human carcinoma cell line A549 cells. In Figure 1, untreated A549 cells (a) and A549 cells treated with TNF-a (2 ng/ml) and IL-1b (10 pg/ml) for 1 hour (b) were trypsinized and probed for NF-B expression and nuclear morphology. Briefly, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% triton, and incubated with mouse anti-NF-B (p65) + Alexa Fluor 488 donkey anti-mouse IgG. Cells were washed and resuspended in 1% paraformaldehyde containing 7-AAD, then mixed with an equal volume of antifade and visualized on slides us-

ing a Nikon Eclipse E600 fluorescence microscope equipped with bandpass filters appropriate for FITC (535/40 nm) and 7-AAD (630/60 nm) fluorescence. NFkB images in grey are depicted on the

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left. NF-B(green) / 7-AAD (red) composite images on the right demonstrate the nuclear localization of NF-B following TNF-a / IL-1b treatment. Visualization of NFkB Translocation in A549 Cells with the ImageStream system Untreated (a) and TNF-a / IL-1b treated (b) A549 cells were prepared as described in Figure 1 and imaged in flow using the ImageStream instrument. Results are shown in Figure 2. Hydrodynamically focused cells were illuminated from the side with a 488 nm laser and from behind with a brightfield source that was passed through a 577/10 bandpass filter. Four out of a possible six images were used in this experiment. Laser side scatter (pseudocolored blue), NF-B fluorescence (green), brightfield, and 7-AAD fluorescence (red) from the cells were spectrally decomposed and imaged simultaneously onto spatially separated channels on a CCD detector. NF-B (green) / 7-AAD (red) composite images on the right demonstrate the nuclear localization of NF-B following TNF-a / IL-1b treatment. Quantitation of NFkB Nuclear Translocation Using Image Correlation Analysis In order to quantitate the extent of NF-B nuclear localization, we analyzed the degree of pixel intensity correlation between the NF-B and nuclear images of each cell within a masked region of interest. Results are shown in Figure 3. The NF-B image of a cell with a high degree of translocation will look qualitatively similar to the nuclear image of that cell, resulting in a high degree of correlation between the two images while the NF-B image of cell without any translocation will have little signal in the nuclear space, resulting in an inverse correlation with the nuclear image. (A) shows the masked areas (turquoise overlay) used for the correlation analysis on an untranslocated and a translocated cell. Two features, the correlation coefficient (r) and a logarithmic transformation of r (similarity) were calculated, and are represented by the following formulas: r= Similarity =

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The variable r measures the degree to which the spatial distribution of intensities over two separate images is correlated, with a range from 1 (inverse correlation) to +1 (complete correlation). The similarity value ranges from to +, allowing standard statistical comparisons (means and standard deviations) between groups to be made. Histogram overlays of NFkB / 7-AAD correlation (b) and similarity (c) distinguish untreated (green) from TNF-a / IL-1b treated (red) A549 cells.

Visualization of NFkB Nuclear Translocation in THP-1 Cells Using Immunofluorescence Microscopy LPS stimulation initiates a signaling cascade that results in the translocation of NF-B from the cytoplasm to the nucleus of the non-adherent human monocyte cell line THP-1. In this experiment (Figure 4), untreated THP-1 cells (a) and THP-1 cells treated with LPS (100 ng/ml) for 1 hour (b) were probed for NF-B expression and nuclear morphol-

Figure 5

Untreated: NF-KB Peak Intensity

Region Single Cells Low similarity Med. similarity High Similarity Count 2760 2638 101 20 %Total 98.3 94 3.6 0.71 %Gated 100 95.6 3.66 0.72 Mean 517.99 517.15 515.04 665.8 Median 513 513 510 654.8 Std Dev 79.75 76.87 91.38 158.69

Untreated: NF-KB / 7AAD Similarity

Region Single Cells Low similarity Med. similarity High Similarity Count 2760 2638 101 20 %Total 98.3 94 3.6 0.71 %Gated 100 95.6 3.66 0.72 Mean -1.246 -1.361 0.881 2.944 Median -1.358 -1.3878 -8.001 2.978 Std Dev 0.844 0.637 0.520 0.345

LPS treated: NF-KB Peak Intensity

Region Single Cells Low similarity Med. similarity High Similarity Count 3695 925 1061 1697 %Total 97 24.3 27.9 44.6 %Gated 100 25 28.7 45.9 Mean 673.26 620.55 600.23 748.04 Median 656 617 599 745 Std Dev 124.52 90.66 85.56 119.38

LPS treated: NF-KB / 7AAD Similarity

Region Single Cells Low similarity Med. similarity High Similarity Count 3695 925 1061 1697 %Total 97 24.3 27.9 44.6 %Gated 100 25 28.7 45.9 Mean 1.7114 -0.804 1.307 3.328 Median 2.114 -0.765 1.291 3.349 Std Dev 1.775 0.644 0.611 0.479

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ogy. Briefly, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% triton, and incubated with mouse anti-NF-B (p65) + Alexa Fluor 488 donkey anti-mouse IgG. Cells were washed and resuspended in 1% paraformaldehyde containing 7AAD, then mixed with an equal volume of antifade and visualized on slides using a Nikon Eclipse E600 fluorescence microscope equipped with bandpass filters appropriate for FITC (535/40 nm) and 7AAD (630/60 nm) fluorescence. Note the relatively thin band of cytoplasm in the untranslocated NFkB images characteristic of this monocytic cell line. Visualization and Quantitation of NFkB Nuclear Translocation in THP-1 Cells Using ImageStream Multispectral Imagery Finally, the translocation of NF-B in THP-1 cells was examined using the ImageStream multispectral imaging technology. Results of the analysis are shown in Figure 5. Untreated (a) and LPS treated (b) THP-1 cells were prepared as described in Fig-

ure 4 and imaged in flow using the ImageStream platform. Plotting NF-B / 7-AAD Similarity vs. NF-B Peak Intensity revealed a range of cells based on similarity that were gated into three populations. Each gated population has an associated image gallery that can be visually inspected. The image galleries here display the NF-B image adjacent to a composite image (green NF-B / red 7-AAD) for each object within a given population. Morphologic inspection of the image galleries revealed that NFkB had translocated to the nucleus in High Similarity but not Low Similarity cells. Medium Similarity cells had a diffuse NF-B staining pattern. Untranslocated and translocated cells had significantly different Similarity values (untreated: -1.36 +/- 0.84 and 2.98 +/- 0.35, respectively; LPS treated: -0.76 +/0.64 and 3.35 +/- 0.48, respectively). Treatment with LPS significantly increased the percentage of NFB-translocated (0.71%, median similarity value 2.98 +/- 0.35 to 44.6%, median similarity value 3.35 +/0.48) and Medium Similarity (3.6% to 27.6%) cells.

Conclusions
This study demonstrated the ability of the ImageStream multispectral imaging cytometer to quantitate the extent of NF-B nuclear translocation in adherent (A549 in response to TNF-a and IL-1b) and non-adherent (THP-1 in response to LPS) cell lines. Images taken using immunofluorescence microscopy (40x objective) and with the Imagestream were qualitatively indistinguishable. The extent of NF-B nuclear localization was quantified using the feature NF-B / 7-AAD Similarity, a logarithmic transformation of the pixel correlation coefficient (r) computed between the NF-B image and a 7-AAD nuclear reference image. By providing population-specific statistical analysis, both the percentage of cells that responded to treatment as well as the extent of response were examined. NFB translocated to the nucleus in nearly all of the A549 cells that were treated with TNF-a and IL1b. On the other hand, NF-B translocated to the nucleus in a subpopulation of THP-1 cells after 1 hour of LPS treatment. Furthermore, the fidelity of these classifiers could be validated by inspection of image galleries corresponding to the gated populations. By providing morphology-based information in addition to fluorescence and laser scatter signals, the ImageStream platform allows for discrimination of cells not feasible with standard flow cytometry.

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