May 1, 2011 (Vol. 31, No. 9)
Claire Bungard, Ph.D.
Xiao Zhang, Ph.D.
Alasdair Robertson, Ph.D.
Multipurpose Solution Aims to Offer Accurate and Quantitative Results
Conventional cell- and bead-based assays can be time-consuming, manual procedures providing only semi-quantitative data. Using high-content analysis systems to obtain object-based, quantitative data is costly and requires expert user input and analysis as well as application-specific modifications.
The CellReporter system from Genetix is a multi-application solution that overcomes these challenges to provide accurate, object-based data suitable for a wide range of assays to determine, for example, cytotoxicity, cell viability, apoptosis, cell proliferation, and antibody quantification. Since assays require no further interaction once set up, the system can be easily integrated into automated robotic systems to increase productivity.
Utilizing state-of-the-art imaging technology and image-analysis software, the system generates high-quality, quantitative data by imaging, analyzing, and generating a report based on individual cells or beads.
The system can be used to run a wide range of assays without the need to change hardware or software. System control and image-analysis software, developed in collaboration with users, provides an intuitive, step-by-step workflow that requires only minimal training. In addition, imaging using multiple wavelength filters makes it possible to multiplex a number of assays, thereby greatly improving speed and output of results.
With many labs under pressure to increase throughput, conventional ELISA assays used to quantify binding and efficacy of different antibodies and antigens become a rate-limiting step. The CellReporter system is ideally suited to run homogenous, bead-based assays used, for example, to quantify antibody production during cell-line development.
The bead-based approach enables a one-step mix-and-read assay as opposed to a multistep multiwash, required for an ELISA. Figure 1A gives an overview of the assay principle: beads are coated with appropriate capture antibody, mixed with antigen (for example, supernatant from cells transfected or fused to produce a specific antibody or protein) and a second fluorescently labelled detection antibody. After complex formation, beads settle at the bottom of the well where they can be imaged individually by the system to produce object-based data.
Figure 1B shows typical images from an IgG quantification assay. During image analysis, a white light algorithm identifies individual beads so that subsequent fluorescence readings relate only to identified objects. This prevents any labeled antibody remaining in solution from distorting results. Fluorescent aggregates will also be excluded from the data thus reducing the risk of false positives—a recognized problem when using conventional plate readers. The mean fluorescence per well is used in the same way as ELISA readings to produce quantitative data from a standard curve to determine antigen concentrations and/ or antibody efficiency.
Quantifying Cellular Responses
The CellReporter system not only performs a wide range of cell-based assays but also enables the study of different parameters of a particular response. For example, the combined use of Hoechst 33342 dye and TxRed labeled antibody against Annexin V enables identification of individual cells and differentiation between apoptotic and nonapoptotic cells after treatment with a range of concentrations of a pro-apoptotic drug.
Using a gating software feature, the proportion of cells that are apoptotic can be quantitatively measured in each well and the efficacy of the drug determined.
When a cell is in the late stage of apoptosis, the nucleus condenses and fragments—revealed by the Hoechst 33342 nuclear dye. The CellReporter system has an advantage over many imaging systems in that it not only detects a signal, but also measures the standard deviation (SD) of the signal within the identified object—in this case the nucleus of the cell. This parameter can be used to separate apoptotic from nonapoptotic cells. The SD of the nuclear stain is high in fragmented nuclei. Figure 2 shows nuclear staining in apoptotic and nonapoptotic cells, and the analysis that reveals the relative proportions of each in a histogram format.
A third parameter, using labeled antibodies against different caspases, can also be studied enabling interrogation of the complete activation chain from onset of apoptosis to cell death.
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.
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.
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.
Claire Bungard, Ph.D. ([email protected]), is application specialist, Xiao Zhang, Ph.D., is R&D scientist, and Alasdair Robertson, Ph.D., is biology discipline leader at Genetix.