Microscopic Analysis of Lysosomal Membrane Permeabilization
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.
Key wordsLysosomal membrane permeabilization Confocal microscopy Image analysis Immunocytochemistry Cathepsin B LAMP-2
This work was supported by grants from the Swedish Research Council, Swedish Cancer Society, County Council of Östergötland, Konung Gustav V och Drottning Victorias Frimurarestiftelse and Stiftelsen Olle Engkvist Byggmästare.
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