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May 1, 2011 (Vol. 31, No. 9)

Analyzing Cell- and Bead-Based Assays

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.

  • Mix-and-Read Assays

    Click Image To Enlarge +
    Figure 1. Homogenous bead-based assay: (A) Schematic showing the principle of the bead assay. The beads are coated with a suitable capture antibody; this is then mixed with the antigen (in this example human IgG) and a suitable fluorescently labelled detection antibody. After incubation, the bead-associated fluorescence can be measured using the CellReporter. (B) Images in the FITC channel of beads coated to capture human IgG: A dilution series of IgG concentration was incubated with the beads, as well as a FITC-labelled capture antibody. The mean FITC per well can be used to produce a reference curve. The amount of antigen in solution can then be calculated for other samples. Keeping the antigen concentration the same and changing the capture antibody can determine relative efficacies of different antibodies.

    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

    Click Image To Enlarge +
    Figure 2. Image and analysis of Hoechst nuclear staining to identify apoptotic cells: (A) Image from a control well, with low proportions of apoptotic cells. (B) Image from a well treated with high concentrations of Staurosporine, a pro-apoptotic agent, with high proportions of apoptotic cells. (C) Histogram showing the number of objects (cells) against the SD of signal within the DAPI channel. When a cell is in a late stage of apoptosis, the nucleus condenses and fragments. The SD of the nuclear stain is then high. This parameter can then be used to separate apoptotic from nonapoptotic cells.

    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.



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