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Microscopic Analysis of Lysosomal Membrane Permeabilization
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.ORCID iD: 0000-0003-4075-159X
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
2017 (English)In: Lysosomes: Methods and Protocols / [ed] Karin Öllinger; Hanna Appelqvist, Humana Press, 2017, Vol. 1594, p. 73-92Chapter in book (Refereed)
Abstract [en]

Lysosomes and lysosomal proteases have been found to participate during several forms of cell death pathways including apoptosis. A critical step in the mediation of apoptotic signaling is the release of cathepsins to the cytosol, a process known as lysosomal membrane permeabilization (LMP). In this chapter, we describe immunofluorescence detection of LMP in cell cultures stained for cathepsin B and LAMP-2 using three confocal techniques namely laser scanning, spinning disk, and aperture correlation spinning disk confocal to obtain images. Image analysis is performed using Huygens software for deconvolution. LMP results in a decrease in the fraction of cathepsin B colocalizing with LAMP-2, which is quantified through Manders colocalization coefficient. Analysis of the images obtained by the three techniques show the same trend but the magnitude of the decrease differs due to the axial resolution. The observations emphasize the use of highest possible resolution when determining colocalization.

Place, publisher, year, edition, pages
Humana Press, 2017. Vol. 1594, p. 73-92
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 1594
Keywords [en]
Cathepsin B; Confocal microscopy; Image analysis; Immunocytochemistry; LAMP-2; Lysosomal membrane permeabilization
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:liu:diva-145060DOI: 10.1007/978-1-4939-6934-0_5PubMedID: 28456977ISBN: 978-1-4939-6932-6 (print)ISBN: 978-1-4939-6934-0 (electronic)OAI: oai:DiVA.org:liu-145060DiVA, id: diva2:1181698
Available from: 2018-02-09 Created: 2018-02-09 Last updated: 2018-02-09

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Villamil Giraldo, Ana MariaÖllinger, KarinLoitto, Vesa
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Division of Cell BiologyFaculty of Medicine and Health SciencesDepartment of Clinical Pathology and Clinical GeneticsDivision of Microbiology and Molecular Medicine
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