Computational protocol: The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis

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Protocol publication

[…] Quantitation of fluorescence was performed using ImageJ Color Profiler software (National Institutes of Health) on selected linear regions in a merge z projection of maximum intensities of 8-bit stacks acquired as described in the previous paragraph with an inverted wide-field microscope (DMB; Leica) equipped with an oil immersion objective (100× Plan Apochromat HCX; 1.4 NA) and a cooled charge coupled device camera (MicroMAX; Princeton Instruments). Two areas of the cell were quantified: the phagocytic cup analyzed as three linear regions and the cell body analyzed as two linear regions. The maximum primary fluorescence intensities of each area were background corrected by subtracting the maximum value from a cell-free region. Ratios were obtained by dividing the maximum of fluorescence intensities in the phagocytic cups by the maximum fluorescence intensities in the cell body and were calculated for F-actin, mDia1, and Arp2/3 complex. [...] Immunofluorescence was performed as described previously () except for staining of EB1 and CLIP-170, which was adapted from . In brief, the cells were fixed for 10 min in ethanol at −20°C, and then in 4% paraformaldehyde for 15 min at RT and permeabilized in 0.15% Triton X-100/PBS. Image acquisition and deconvolution were performed as described previously (), except that the samples were examined under an inverted wide-field microscope (Axiovert 100M [Carl Zeiss, Inc.] or DMB) equipped with an oil immersion objective (100× Plan Apochromat HCX; 1.3 or 1.4 NA, respectively) and a cooled charge coupled device camera (ORCA ER [Hamamatsu Photonics] or MicroMAX, respectively). Z series of images were taken at 0.2-μm increments. 3D reconstructions were obtained using the IsoSurface function of Imaris 5.7 software (Bitplane AG). To quantitatively evaluate the level of colocalization, the Pearson's correlation coefficient () was calculated, with a value of one indicating complete positive correlation and zero indicating no correlation. All values were obtained with the plug-in JACoP (), developed for the image analysis software ImageJ. Each pairwise comparison was done on a z plane acquired with a 0.2-μm step and divided in two regions: a phagocytosing and a nonphagocytosing region. […]

Pipeline specifications

Software tools ImageJ, Imaris, JACoP
Applications SPIM, Microscopic phenotype analysis