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Cell-Death and Autophagy

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Poster:
In vitro assay of early cell dysfunction using multispectral image-in-flow cytometry of mitochondrial morphology in a EFGP-expressing cell line		   Technical Information:
Assessing Autophagy with the FlowSight® Imaging Flow Cytometer
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Merck:/Freestyle/BI-Bioscience/Cell-Analysis/amnis/Cell-death-100x100.jpgThe Amnis® imaging flow cytometry system is advancing the study of cell death and survival. Morphological characterization by microscopy remains the gold standard for accurately identifying the various types of cell death using characteristics such as nuclear condensation, nuclear fragmentation, membrane blebbing, cell shrinkage or swelling. In some cases cell death is preceded by an attempt at survival by autophagy and is identified by the clustering of the phagolysosome membrane-associated protein LC3. Combining the measurements of cell death with immunophenotyping or other Amnis® imaging flow cytometry applications such as signal transduction increases the power of your experiments.

Featured Video

Watch to learn how multispectral imaging in flow can be used to enhance apoptosis research. Dr. Sherree Friend explains how Amnis® applications use high-throughput imaging of DNA fragmentation in apoptotic cells. Assessment of the process of cell death is revolutionized by analyzing visual characteristics in thousands of cells.

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Apoptotic Index Using the ImageStream®X

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Dramatic changes in nuclear morphology are hallmarks of apoptosis. When cells begin to die by apoptosis there is fragmentation and condensation of the DNA. This makes possible the automated identification of apoptotic cells. By measuring the area and the intensities of the brightest portions of the nuclear image, the bright, punctate nuclear imagery of apoptotic cells can be distinguished from the evenly stained nuclear imagery of a normal, healthy nucleus.

Autophagy Measurement on the ImageStream®X

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Autophagy, the process of degrading a cell's own components through the lysosomal machinery in response to stress, plays a normal role in cell growth, development and homeostasis. During autophagy, the microtubule associated protein LC3 is recruited to the membrane of autophagosomes and can be visualized as clusters using immunofluorescence microscopy. Here we show the ability to quantify autophagy using a spot count feature which enumerates the bright, punctate spots of GFP-LC3. The quantitative nature of the image data allows detection of autophagy even in rare subpopulations of cells and enables the automated identification of cells undergoing autophagy.
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