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Analyzing Cell- and Bead-Based Assays

Multipurpose Solution Aims to Offer Accurate and Quantitative Results

• Claire Bungard, Ph.D.
• ,
• Xiao Zhang, Ph.D.
• ,
• Alasdair Robertson, Ph.D.

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Details of Cellular Responses

The system can be used to measure any stable fluorescent signal down to a resolution of 0.8 µm. However, certain organelles that cannot be resolved such as mitochondria, can be analyzed regardless. Analysis can be done using TMRE accumulation as an indicator of mitochondrial activation. Although individual mitochondria cannot be clearly resolved, the fluorescence around the nucleus of cells containing active mitochondria can be measured. This enables a rapid and quantitative measurement of active mitochondria.

Following Translocation

Figure 3. Image and analysis of NFkappaB translocation: Stimulated and unstimulated cells were incubated with a Cy3 labeled anti-NFkappaB antibody. In unstimulated cells the NFkappaB remains in the cytoplasm only (A); upon stimulation it is translocated from the cytoplasm into the nucleus (B). Within the statistics manager area of the CellReporter software, a ratio of interior to exterior can be defined. When this ratio is 1 or above, the NFkappaB is equal or greater in the nucleus than the cytoplasm, and therefore, the cells are stimulated, as shown in (C). Click Image To Enlarge +

Figure 3. Image and analysis of NFkappaB translocation: Stimulated and unstimulated cells were incubated with a Cy3 labeled anti-NFkappaB antibody. In unstimulated cells the NFkappaB remains in the cytoplasm only (A); upon stimulation it is translocated from the cytoplasm into the nucleus (B). Within the statistics manager area of the CellReporter software, a ratio of interior to exterior can be defined. When this ratio is 1 or above, the NFkappaB is equal or greater in the nucleus than the cytoplasm, and therefore, the cells are stimulated, as shown in (C).

The CellReporter analysis tools are well suited for translocation assays. The system takes fluorescent readings from within and around the nucleus then allows the user to create their own statistics within the software. Figure 3 gives an example of imaging and analysis from an NFkappaB translocation assay. Here the ratio of staining from the cytoplasm (exterior) to the nucleus (interior) is created as a statistic. As the ratio moves to 1 or above, the proportion of activated cells can be quantified.

Conclusion

The examples presented here illustrate the application flexibility of the CellReporter system. The rapid, high-resolution imaging and advanced image analysis provide accurate, quantitative results for a wide range of cell-and bead-based assays overcoming the challenges presented by conventional techniques.