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Untreated (non-apoptotic) MCF-7 cells imaged under white light
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Untreated MCF-7 cells imaged using fluorescence (DAPI)
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Data analysis
- Mean fluorescence used to gate cells into three populations - viable (blue), apoptopic (yellow) and dead cells (pink)
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Untreated cells
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All cells
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Review individual cells in high resolution images for each fluorescent channel
User defined analysis in the scatter plots is represented in the images
Report
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Plate Overview shows number of cells and proportion of apoptotic cells per well
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Assay principle:
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Apoptosis induced by Anisomycin
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Hoechst 33342 binds to DNA of all cell nuclei
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Propidium iodide enters only dead cells
- Fluorescent Annexin V binds to altered membrane of apoptotic cells
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Apoptosis induced by Anisomycin
Further information
- Screening and quantification of apoptosis induction
Cellular responses - assess apoptosis through morphology
Assessment of apoptosis through changes in cell nuclear morphology
Image acquisition, accurate identification of nuclei
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Untreated (non-apoptotic) HeLa cells
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Treated (apoptotic) HeLa cells showing condensed, fragmented chromatin
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Data analysis
- Gating of non-apoptotic and apoptotic populations
Histogram: distribution of standard deviations of nuclear intensities in DAPI channel.
Analysis within histogram is represented in the images.
Analysis within histogram is represented in the images.
Report
- Plate Overview reveals distribution of apoptotic cells per well
Diameter of pie chart = cell number
Slices in pie chart = apoptotic:non-apoptotic cells
Slices in pie chart = apoptotic:non-apoptotic cells
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Non-apoptotic cell with decondensed, non-fragmented nucleus
Apoptotic cell with condensed, fragmented nucleus
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Further information
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Assessment of apoptosis through changes in cell nuclear morphology
Cytotoxic response - determine 'dead-cell' population
Assessment of ‘dead-cell’ dye uptake following cytotoxic challenge
Image acquisition, accurate identification of nuclear and cytoplasmic regions
Nucleus of all cells stained with Hoechst 33342
Nucleus of dead cells stained with propidium iodide
Data analysis
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Cell population gated into sub-groups: live / dead
Histogram plot: distribution of nuclear fluorescence in Texas Red channel
Report and export data
- Plate Overview reveals distribution of dead cells within each well
- Export to create a dose response curve
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Diameter of pie chart = cell number
Slices in pie chart = live:dead cells |
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Dose response curve from exported data
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Further information
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Assessment of ‘dead-cell’ dye uptake following cytotoxic challenge
Cell viability - determine 'live-cell' population
Assessment of cytoplasmic ‘live-cell’ dye hydrolysis following cytotoxic challenge
Image acquisition, accurate identification of nuclear and cytoplasmic regions
Nucleus of all cells stained with Hoechst 33342
Fluorescent (viable) cells
Data analysis
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Cell population gated into sub-groups: viable/non-viable
Histogram plot shows distribution of cytoplasmic fluorescence in the FITC channel
Report and export data
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Plate Overview reveals distribution of cell viability across plate
- Export to create a dose response curve
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Diameter of pie chart = cell number
Slices in pie chart = viable:non-viable cells |
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Dose response curve from exported data
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Further information
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Assessment of cytoplasmic ‘live-cell’ dye hydrolysis following cytotoxic challenge
Cellular activation state - determine protein localization
Assessment of NF-ĸß protein localization
Image acquisition and accurate identification of nuclear and cytoplasmic regions
Nucleus of all cells stained with Hoechst 33342
All cells stained with anti – NF- ĸß antibody revealing NF- ĸß localization
Data analysis
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Determine ratio of nuclear:cytoplasmic fluorescence - gating of stimulated and unstimulated cells
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Histogram: distribution of Cy3 interior mean fluorescence: Cy3 exterior mean fluorescence. |
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Fixed HeLa cells. Analysis within histogram is represented in the images.
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Report
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Plate Overview reveals distribution of stimulated cells per well
Diameter of pie chart = cell number
Slices in pie chart = stimulated:unstimulated cells
Slices in pie chart = stimulated:unstimulated cells
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Further information
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Assessment of NF-ĸß protein localization
Cell proliferation - determine cell cycle profile
Assessment of cell cycle via changes in nuclear DNA content
Image acquisition, accurate identification of nuclei
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Fluorescent imaging
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Nuclei identified
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Data analysis
- Heterogeneous cell population gated into 3 sub-groups
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Histogram: distribution of total nuclear intensities in Texas Red channel |
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Analysis within histogram represented in images |
- Gating of sub-groups further sub-divides cell population
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G2 cells (4n DNA, de-condensed nuclei) distinguished from pre-anaphase mitotic cells (4n DNA, condensed nuclei)
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Post-metaphase mitotic cells (2n DNA, condensed nuclei) distinguished from G1 cells (2n DNA, de-condensed nuclei)
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Report
- Plate Overview reveals cell cycle distribution of cells within each well
Diameter of pie chart = cell number
Slices in pie chart = proportion of cells in each cell cycle phase
Slices in pie chart = proportion of cells in each cell cycle phase
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Changes in nuclear DNA throughout the cell cycle
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Further information
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Assessment of cell cycle via changes in nuclear DNA content
Bead-based assays
In comparison with conventional techniques, the CellReporter system supports a broader range of applications utilizing the bead-based assay format. The assay workflow is also less time-consuming, requiring only a single incubation step, no wash steps and a simple, intuitive workflow to screen, analyze, visualize and export data.
Antibody quantification - comparison of bead-based assays to conventional ELISA
Quantification of IgG production
Time-saving, simplified workflow
Replace time-consuming ELISA assay with one easy incubation step (no washing required)
Image acquisition, accurate identification of beads
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Wells are imaged
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Beads are identified
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Accurate overlay for bead-specific quantification of IgG
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Data analysis
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Plate Overview illustrates a heat map representation of the distribution of fluorescent bead intensity per well across the plate
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Report
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Standard curve shows comparable results between the homogeneous bead assay and an ELISA approach
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Conclusion
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IgG levels were accurately quantified by identification of individual beads and determination of their fluorescence
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The sensitivity and dynamic range of this approach is comparable to conventional ELISA but saves time - only a single incubation step and no wash steps are required
Further information
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Comparison of a homogeneous bead-based assay format with conventional ELISA
Brochure
CellReporter™
Quantification of cellular responses
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Recent publications
Technical note: Analyzing Cell- and Bead-Based Assays
GEN Tech Notes: May 1, 2011 (Vol. 31, No. 9)
Paper that demonstrates how CellReporter offers a multipurpose solution for accurate and quantitative results for cell- and bead-based assays.
Recorded webinars
Phenotypic Screening to Identify Cures for the Deadliest Poison
Dr. Tobin Dickerson, The Scripps Research Institute, San Diego, CA
Dr Dickerson discusses the development of a whole cell based assay for Botulinum neurotoxin (BoNT) inhibitors based on the new CellReporter™ multi-application platform. Listen now >
Accelerated high content screening and homogeneous quantification of soluble IgG levels
Dr Alasdair Robertson, Head of Biology R&D, Genetix Limited
Dr Robertson demonstrates how utilizing the CellReporter™ system's quantitative approach to assessing IgG levels can save time and simplify workflow compared to conventional methods such as FMAT®.
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