Computational protocol: Abnormal Processing of Autophagosomes in Transformed B Lymphocytes from SCARB2-Deficient Subjects

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Protocol publication

[…] Colocalization of fluorescence of GFP and GRP78BiP in Cos7 cells was quantified using the software NIH ImageJ 1.34 (http://rsbweb.nih.gov/ij). The software was used to measure the area of pixels of SCARB2 (GFP in the green channel) and GRP78BiP (red channel) after adjustment of the threshold to minimize background noise while retaining the signal. This process was performed in the same manner for all analysis of transfected Cos7 samples by the same operator. Using JACoP (http://rsbweb.nih.gov/ij/plugins/track/jacop.html), a plugin for the software ImageJ, the Manders' coefficient, as a measure of the ratio of the “area of pixels of GFP colocalizing with GRP78BiP” to that of the total GFP, for each cell was generated. All images were acquired under the same settings, in particular the same pinhole for an identical section thickness of 0.8 μm, by the Zeiss LSM 510 Meta confocal microscope (Göttingen, Germany). Images were analyzed by z-series to ensure that the areas of colocalization existed in all three dimensions and not due to pixels from two different channels at various levels of the sections. […]

Pipeline specifications

Software tools ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Human gammaherpesvirus 4
Diseases Renal Insufficiency