Live Cell Imaging

Gen5 Well Mode Imaging combined with Reagent Injector Module allows for Visualization and Quantification of Rapid Kinetic Changes

Kinetic imaging requiring a fast frame rate, such as Ion Channel Assays, should be performed using Gen5 Well Mode kinetic imaging and the reagent injector module. This set-up allows for images to be rapidly captured in a single well after reagent addition before moving to the next well to perform reagent injection and imaging. Figure 1 below shows a well-mode kinetic imaging protocol set up with injection (dispense) in the Gen5 software. The kinetic imaging and injection steps are defined inside the ‘Well Mode’ brackets.

• Be mindful of the integration time (exposure time) set for the imaging channel when looking to image rapid kinetic changes. Decreasing the integration time and turning on ‘capture binning’ allows for faster frame rate imaging.
• When assessing a rapid change after reagent injection, it is important to acquire images before reagent injection to establish a baseline (see Figure 1).

To set up well mode kinetic imaging, ‘Well Mode’ is selected under the ‘Process Mode’ in the Gen5 procedure window. Then, ‘Start Kinetic’ is used to define two kinetic imaging reads, one shorter one before the reagent dispense step, to establish a baseline, and one after the dispense step to monitor changes. The data from the second imaging step is automatically appended to the first imaging step.

• When analyzing kinetic data in Gen5, data can be automatically normalized to the baseline acquired before reagent injection. After deciding how all images are to be analyzed, using either cellular analysis or image statistics, a kinetic analysis step can be defined in the Gen5 data reduction window (see Figure 2A). This allows for the average signal from baseline images to be easily calculated. Once this step is added, a ‘Ratio Transformation’ step can be performed (see Figure 2B) for automated normalization of all data points to the baseline calculated in the kinetic analysis step.

To normalize data, such as fluorescence after reagent injection, to the baseline signal before reagent injection, a kinetic analysis step should be performed inside Gen5 (A). Using a kinetic analysis step, the software can automatically calculate the average signal from all images captured during the baseline imaging (before reagent injection). This software calculated data output (mean baseline) is then used in a ‘Ratio Transformation’ step to normalize all data (B), and curves of this normalized data are automatically displayed in the software (C).

Long-term live cell imaging

Fluorescent nuclear markers

When performing live cell imaging assays, it is crucial to consider the overall viability of your biological sample. Hoechst is a commonly used nuclear marker that binds into the minor groove of A-T rich regions of DNA. Because it readily passes through both the plasma membrane and nuclear envelope, it is an ideal fluorescent dye for live-cell imaging. However, it is important to keep in mind the following:

• At high concentrations, Hoechst 33342 inhibits proliferation rates (Figure 4A), which correlates with increased necrotic cell death, as assessed using propidium iodide (not shown).
• Recommendation: when supplemented in the culture medium during a kinetic read, we recommend using it at or below 100 nM (~60 ng/mL).
• Caution: using Hoechst at low concentrations ensures cell viability, however attaining sufficient signal requires an extended incubation time (3-4 hours), and the Signal-to-Noise Ratio (SNR) might not be as robust as ideal. Sustained exposure to high-energy UV light via increased LED intensity or integration time can have phototoxic effects. Therefore, adjusting Digital Gain first may be a suitable alternative before adjusting these other capture settings.
• Use of a far red-shifted nuclear marker, such as DRAQ5, which is imaged using the CY5 imaging channel, can also help overcome the negative effects of Hoechst 33342, as probes imaged in the red spectral range have far less phototoxic effects over time.

Normalizing cell signals to account for cell proliferation during long-term imaging

The High Contrast Brightfield (HCBF) cell counting method uses transmitted light and specialized optics to achieve accurate cell counts as an alternative to fluorescent live-cell stains. This method is available for both the Lionheart and Cytation imager instruments and can readily be employed to monitor cell proliferation during long-term live-imaging assays.

• Recommendation: Some cell types are less amenable to the HCBF cell counting method due to their morphological phenotype, such as highly clustered cell types. It is necessary to establish HCBF parameters for your cell type of interest.
• Caution: Accurate cell counts are essential for the correct representation of long-term imaging assay results. However, cell counts based solely on the fluorescent signal representing a treatment effect can be vulnerable to misinterpretation.

As an example, Figure 4 represents an application where fluorescent dyes were used to evaluate apoptosis in response to varying concentrations of anti-neoplastic drugs over three days. Apoptotic cell counts were determined by quantifying fluorescently labeled cells stained by the pSIVA apoptosis marker, and total cell counts were determined independently in Gen5 using the HCBF cell counting method. Evaluating the drug effect using only the absolute counts from the fluorescent channel resulted in EC50 values that varied depending on the time point investigated (Figure 4A, lower panel). This erroneous conclusion is corrected using HCBF to account for differences in total cell populations across the different treatments. After normalization, EC50 values remain consistent over time (Figure 4B, lower panel).

Note: Normalization is accomplished automatically in Gen5 by dividing the apoptotic cell counts by the HCBF cell counts and expressing the result as the percentage of apoptotic cells.

Webinars OnDemand

• A Practical Guide for 3D Cell Culture Systems and Optimizing Spheroid Imaging Assay - Part 1
• A Practical Guide for 3D Cell Culture Systems and Optimizing Spheroid Imaging Assay - Part 2
• Cytation C10 Confocal Imaging Reader: A Overview of the Unique Combination of Imaging and Detection Modes that Support a Broad Range of Cell Analysis Applications
• Techniques for Improving Cell Culture Quality Control and Assay Optimization

Application Compendiums

• Automated Methods for Conducting Cell Culture Quality Control Studies and Assay Optimization
• Live Cell Proliferation and Viability
• Kinetic Fluorescence Live Cell Assays