Analysis parameters Provides statistical analysis of cell populations via fluorescence intensity.
Measures fluorescence intensity, morphology, localization, and movement. Time-lapse experiments. Data can be exported in FCS file format.
Detection mode Provides single-cell resolution using PMT detection. Provides subcellular resolution using a CCD camera.
Cell and plate throughput Rapid analysis of thousands to hundreds of thousands of cells per tube/microplate well. One to approximately 60 microwell plates per day, manual operation.
Rapid analysis of one to thousands of cells per micoplate well. One to approximately 200 microwell plates per day, automated operation.
Cell preparation Cells must be prepared in suspension. Also compatible with multiplexed bead arrays.
Ideally suited to adherent cells and tissues, but cells in suspension can be analyzed.
Number of channels Multiplexible (12+ colors). Multiplexible (4+ colors).
Sample Utilization Cells are normally discarded but can be collected following sorting.
Live cells in culture can be revisited and monitored over time, prepared plates can be reimaged as needed.
Key strengths • Statistical population analysis
• Cell preparation (sorting)
• Detection of rare events
• Visual confirmation of data
• Spatial analysis
• Morphological measurements
Do you need temporal and spatial information?
Do you need to measure cell or colony morphology parameters?
Do you work with adherent cells?
Do you have low numbers of cells to analyze?
Do you need to make live-cell kinetic measurements?
Is sample throughput holding you back?
Does reducing large data sets to meaningful outputs take too long?
If so, high-content imaging can compliment your flow-based techniques.
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Flow Cytometry and High-Content Imaging are ComplimentaryFlow cytometry and high-content imaging collect and analyze multiplexed data from individual cells. The processes are very similar for both platforms; cells are either analyzed live or fixed (and permeabilized when appropriate). Depending on the experiment, cells, cellular proteins, cellular structures, or subcellular organelles are labeled with fluorescent probes (antibodies, dyes, fluorescent proteins, or nanoparticles) for detection and analysis of biological processes. Also, on both platforms, unlabeled cells can be analyzed for certain parameters.
High-Content Imaging Can Provide Additional Insight into Cellular EventsSpatial Information—Images acquired on BD Pathway™ bioimaging systems are processed by the system’s powerful image analysis software. The first step is to apply segmentation, which delineates regions within each cell where cellular measurements will be obtained. Measurements can be of the whole cell or from one or several subcellular compartments (Figure 1). This type of spatial information provides insight into protein translocation and other cellular trafficking events.
Figure 1. Segmentation. Representative 20x (grayscale) images acquired on a BD Pathway™ 855 were processed using BD AttoVision™ software to illustrate whole cell, nuclear, cytoplasmic, and plasma membrane segmentation. Corresponding segmentation masks (displayed in random color) are shown.
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Kinetic Measurements—Many biological reactions occur too quickly for a timed endpoint formatted experiment to be useful. In addition, during assay development, kinetic experiments are valuable for determining optimal parameters for endpoint experiments.
Figure 3. Kinetic measurements. Cropped 20x intensity-based pseudocolored images (left) are shown from selected timepoints in a kinetic experiment where cells labeled with Fluo-4 calcium dye were sequentially treated with an agonist and a control compound. As displayed by both the images and the kinetic traces of the individual cells (right), cells had a heterogeneous response to the agonist both in intensity and time of response, and a more homogenous response to the control compound. Pseudocolor bar shows intensity range from low (top) to high (bottom). Cells were imaged in kinetic mode on a BD Pathway™ 855 using environmental control.
Figure 2. Morphology. Cells stably expressing BD Pharmingen™ Red FP—Mitochondrion organelle vector (pseudocolored red) were treated with staurosporine to induce apoptosis in time and concentration dependent experiments. Cells were fixed and stained with Hoechst (pseudocolored blue) prior to imaging on a BD Pathway™ 855. Cropped (20x) images show normal morphology in control cells (left) and condensed mitochondria and nuclei in treated cells (right). Morphological measurements for both cytoplasmic and nuclear areas were analyzed and graphed using BD image and data analysis software. BD data analysis software was used to create a heat map and EC50 curve from a concentration dependant experiment.
Morphological Measurements—Cellular events triggered by processes such as cell signaling, cell cycle, apoptosis, and differentiation can have a multitude of effects, including changes in morphology of the cell or subcellular structures. These effects can be quantitatively measured on a BD Pathway™ bioimaging system.
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Flow Cytometry and High-Content Imaging are SynergisticFlow sorting can be used to enrich for specific subpopulations of cells. This application of flow cytometry is beneficial in stem cell research where differentiation of stem cells generally produces mixed cultures. Imaging is ideal for the study of subpopulations postsort, especially when the number of cells isolated may be low or when further growth or differentiation of cells may make analysis by flow cytometry undesirable.
Figure 4. Human ES cells (NIH approved H9 cell line) were used to derive neural stem cells (NSC), which were then differentiated into neurons in 96-well microplates or 10 cm dishes for high-content imaging and flow cytometry, respectively. After one week of culture, cells were processed and labeled for imaging using the following BD Bioimaging Certified directly conjugated antibodies: Alexa Fluor® 647 anti-Sox2 (pseudocolored yellow), Alexa Fluor® 555 anti-Nestin (pseudocolored red), and Alexa Fluor® 488 anti-MAP2B (pseudocolored green); cells were also stained with Hoechst (pseudocolored blue), and then imaged on a BD Pathway™ 4�5 with a 10x objective. As anticipated, a mixed population of NSC (nestin+) and neurons (MAP2Bbright) were detected. Cells that were cultured in the 10 cm plates were dissociated and stained with two BD antibodies, PE-Cy7 anti-CD56 and a PE anti-CD184 prior to being sorted on a BD FACSAria™ II at 70 psi using a 70 micron nozzle. CD56 is a marker for neurons and neural stem cells and CD184dim cells identify differentiated neurons (MAP2Bbright). The CD56+/CD184dim cells were gated on, sorted, and then transferred to a BD Falcon™ 96-well plate. The cells were stained and imaged as above. The staining patterns demonstrate that sorting resulted in enrichment of neurons (MAP2Bbright cells). A scatter plot (generated in BD data analysis software) shows the dramatic reduction of nestin+ cells in the postsort population (black diamonds—presort, red squares—postsort). Additional analysis of the cultured neurons was done using BD Neurite Outgrowth software, the neurite segmentation mask and average neurite length data is shown.
