Computational protocol: Modifications of the endosomal compartment in peripheral blood mononuclear cells and fibroblasts from Alzheimer's disease patients

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Protocol publication

[…] PBMC isolation from blood samples: For each participant, 10 ml blood was collected in heparin-coated tubes (Vacutainer, Beckton-Dickinson, Franklin Lakes, NJ, USA). Blood samples were diluted in one volume of phosphate-buffered saline and PBMCs isolated by centrifugation on a 1077 g ml−1 ficoll gradient (15 ml prefilled filtered Leucosep PANCOLL tubes, PAN Biotech, Aidenbach, Germany) following the manufacturer's instructions. After centrifugation, PBMCs were re-suspended in 200 μl of reduced serum culture medium (Gibco OPTI-MEM) and incubated at 37 °C, 5% CO2 for 1 h, then rinsed and fixed for 12 min in 4% cold paraformaldehyde and rinsed in 10 ml phosphate-buffered saline.Culture of fibroblast: Fibroblasts were grown directly on microscope coverslips in 24-well plates, in DMEM supplemented with 10% FCS and 100 μg ml−1 penicillin+streptomycin, for 24 to 48 h. Cells were fixed before reaching confluence in 4% cold paraformaldehyde for 15 min and rinsed three times with phosphate-buffered saline. The number of passages was between 3 and 12 at the time of analysis.Immunocytochemistry: Paraformaldehyde-fixed PBMCs were incubated with 2% normal goat serum for 20 min and permeabilized 20 min in Triton 0.1% X-100. For staining of EEA1, cells were re-suspended in 1:100 solution of Polyclonal antibody (Cell signalling, Rabbit-anti-Human #2411) in 2% normal goat serum at 5 °C overnight. Cells were rinsed in phosphate-buffered saline twice and incubated with 1:500 Alexa-488®-conjugated Goat-anti-Rabbit antibody (A-11034, Invitrogen) in 2% normal goat serum, for 2 h at room temperature. Slides were mounted by mixing 10 μl of re-suspended cells in 10 μl mounting medium (SouthernBiotech, fluoromount-G) and sealed after one night air drying at room temperature.For fibroblasts, EEA1 immuno-staining was performed following the same protocol directly on coverslips with an additional staining of nuclei before the last wash using DAPI for 2.5 min. Mounting was performed by applying coverslips upside down on a drop of mounting medium and sealed after one night air drying at room temperature.Confocal imaging: Immunofluorescent labelling was observed under an upright confocal microscope (Olympus Fluoview Fv1000) using a × 63 (NA 1.40) apochromatic objective. All PBMC images were performed at the same magnification. (Voxel size for PBMC: XY=0.073 μm, Z=0.25 μm). Due to the large size of fibroblasts, we applied a lower magnification (Voxel size: XY=0.094 μm, Z=0.25 μm). Approximately 20 cells per subject for PBMCs and 15 cells per subject for fibroblasts were chosen randomly on the slides and scanned individually by defining the upper and lower position of the scanning device and by fixing the space between slides to 250 nm (the actual depth of field of the objective) to sample the whole cell volume for subsequent three-dimensional image treatment.Image analysis of the endosomes: The three-dimensional reconstructed images were analysed applying a wavelet-based detection method implemented as a plugin spot detector in Icy software (http://icy.bioimageanalysis.org.) as described earlier for the analysis of cells from DS individuals. The size was expressed as the number of voxels in the detection, and when needed, converted in cubic micrometres (μm3) according to voxel size. Staining and image analysis were performed blindly by the same operator to guarantee anonymous and unbiased interventions at all levels of sample processing and to avoid inter-operator variability.Automated data processing: Data extraction and analysis was automatized using MATLAB version 8.3.0.73043 (R2014a). The function selects relevant data in the single result files provided by Icy software for each cell, and implements a concatenated database of all subjects by classifying them automatically with respect to clinical information, relating every single detected endosome to the cell, as well as to the individual and his clinical information. […]

Pipeline specifications

Software tools Spot Detector, Icy
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Alzheimer Disease, Dementia