Computational protocol: Identification of the phospholipid lysobisphosphatidic acid in the protozoan Entamoeba histolytica: An active molecule in endocytosis

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Protocol publication

[…] Confocal microscopy images were analyzed with Image J 1.48i software . To quantify the fluorescence intensity inside the cell, we used images of maximum projections. The region around each cell was drawn and the cellular area, the integrated intensity and the mean gray values were measured. Measurements of other regions without fluorescence were used for background subtraction. The net average fluorescence intensity per pixel, expressed as corrected total cell fluorescence (CTCF), was calculated for each trophozoite and time point with the formula , :CTCF= Whole cell signal−(area of selected cell × fluorescence of background). Where whole cell signal=sum of pixels intensity for each cell (integrated intensity value). Fluorescence of background=average signal per pixel for a region without fluorescence selected just beside the cell (mean gray value).To quantify stained pinosomes and phagosomes, as well as Lysotracker stained vesicles, 1-μm Z-stacks of whole cell were acquired. To calculate the relative percentages of merging vesicles during pinocytosis, the number of FITC-dextran stained pinosomes was considered as 100% at each time, then, percentages of merging pinosomes were obtained. For quantification of LBPA in phagocytic trophozoites, the number of ingested erythrocytes was taken as 100%, then, the percentage of 6C4 of stained phagosomes was calculated. For triple labeling experiments, 6C4, anti-EhADH, Lysotracker and merging stained vesicles with or without erythrocytes was calculated taking as 100% the 6C4 stained vesicles. To determine co-localization in entire cell or an area around merging vesicles, merging channels of 1-μm Z-stacks (n=150 sections) were contrasted, then, colors were separated to be analyzed using the Just Another Co-localization Plugin (JACoP) in the ImageJ 1.48i software to calculate Pearson´s coefficient (PC). Each point represented an average and values are given as means±standard deviation. […]

Pipeline specifications

Software tools JACoP, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Entamoeba histolytica
Diseases Multiple Sclerosis
Chemicals Fluorescein-5-isothiocyanate