Abstract
Lysosomal DNase II in phagocytic digestion produces DNA ends with 3′PO4/5′OH, which differ from those created in apoptotic DNA fragmentation, and can be used to label phagocytic clearance of cell death. Here, we describe the use of these specific DNA ends as selective markers of phagocytic reaction in cell suspensions. The approach does not require cell fixation. It selectively labels blunt-ended DNA breaks with terminal 5′OH. The detection is performed by ultra-fast FRET probes in a single step, closed-tube procedure. It takes 3 min and is signaled by fluorescence. The full step-by-step protocol is presented as well as instructions on analysis and representation of the results.
The described DNA-end-based phagocytosis marker and the new rapid FRET assay can be useful in studies of phagocytosis, apoptosis and in immune system assessments.
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Acknowledgment
I am grateful to Candace Minchew for her outstanding technical assistance.
This research was supported by grant R01 NS082553 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health and by grants R21 CA178965 from the National Cancer Institute, National Institutes of Health and R21 AR066931 National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health.
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Didenko, V.V. (2017). Express FRET Labeling and Analysis of Phagocytic Clearance. In: Didenko, V. (eds) Fast Detection of DNA Damage. Methods in Molecular Biology, vol 1644. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7187-9_1
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DOI: https://doi.org/10.1007/978-1-4939-7187-9_1
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7187-9
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