| Apoptosis and DNA Damage Applications |
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| Introduction |
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| Apoptosis detection methods are well suited to laser scanning cytometry. In addition to quantitative data relevant to apoptosis, laser scanning cytometry creates images of cells to confirm changes in nuclear morphology. Along with the apoptosis applications developed for the LSC, a number of newer detection methods have been examined with iGeneration systems taking advantage of their automated capabilities. Apoptosis assays for live cells are discussed under Live Cell Studies. |
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| Highlights of Analysis with iGeneration Imaging Cytometers |
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| Recent reagent developments provide tools to examine signal transduction cascades during apoptosis. Two examples are detecting cleaved caspase-3 and the phosphorylation of H2AX. |
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- Caspase-3 is an important protease, activated by cleavage as a step in certain apoptosis-signaling pathways.
- H2AX is a histone phosphorylated at sites of double-stranded DNA breaks, thereby acting as a reporter of double-stranded DNA breakage.
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| In both cases, drug titration is coupled to the detection of these apoptosis markers. |
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| Any number of wells in a multi-well plate may be analyzed: |
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| Phosphorylated H2AX Detection |
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| In the following example, the phosphorylation level of H2AX is plotted per well as a histogram. Three control wells are shown on the right; the nine remaining histograms show the effect of camptothecin. Shown below is a difference test comparing the control data to each well. |
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| Cell Cycle Effects |
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| Analysis of cell cycle effects can be combined with cell cycle analysis by examining DNA content histograms and the difference plots for these data in the same cell population, as shown below. |
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| Specific populations may be viewed for their morphological characteristics. Using data windows, shown below, regions may be drawn and events in that region viewed in a gallery. |
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Analysis can be quantified and reported using either CompuCyte software or third party software.
Phosphorylated H2AX Antibody Staining
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| Cleaved Caspase-3 Studies |
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| Similar analyses may be undertaken for cells identified with a specific probe for cleaved caspase-3. The following fields show images from four wells with control (C1), low (C4), moderate (C7), and high (C12) amounts of an apoptosis-inducing drug. For the same wells we can measure the DNA content (left) and amount of cleaved caspase-3 (right). |
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| Benefits of Using iGeneration Technology |
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| Multiple probes (for example, DNA content, phospho-H2AX and caspase-3 cleavage) can be analyzed in a single sample while obtaining widefield images, which may be correlated to the quantitative data in galleries or field images. The iGeneration platform combines the advantages of flow cytometry and image analysis in a single system. The iGeneration inverted format supports a diversity of carrier types, allowing flexibility in matching the needs of your application with the appropriate carrier type. |
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| More Information |
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For more information, review the CompuCyte Published Materials and CompuCyte Bibliography.
Request the Apoptosis Application Note.
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