Computational protocol: TERRA, hnRNP A1, and DNA-PKcs Interactions at Human Telomeres

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Protocol publication

[…] Images were acquired and analyzed using a Zeiss Axio Imager.Z2 epi-fluorescent microscope running Metamorph software (Molecular Devices). For each cell, 22-stacked images taken at 0.2 μm intervals, per channel, were obtained with a 1.25 numerical aperture 100× oil immersion objective then analyzed by 3D deconvolution and reconstruction. ImageJ software (http://rsb.info.nih.gov./ij/) and JACoP plugin (National Institute of Health; NIH) were used to evaluate co-localization of TRF2 (telomeres) and TERRA foci from 3D images in the red channel for TRF2 (anti-mouse AlexaFluor-594; Life Technologies) and the green channel for TERRA (FITC labeled PNA probe; Bio-Synthesis Inc, Lewisville, TX, USA). Several co-localization coefficients such as Pearson’s, Mander’s, and Overlap were calculated using the ImageJ and JACoP plugin, to assess the degree of signal co-localization in the 3D images and generate co-localization profiles for each individual cell. Additionally, ImageJ software was utilized for co-localization foci counts and evaluation of the foci numbers. Costes randomization was used to exclude any co-localization of pixels that might have occurred due to chance (Bolte and Cordelieres, ). For each condition, 40 TERRA positive cells were imaged (n = 40). […]

Pipeline specifications

Software tools MetaMorph, ImageJ, JACoP
Application Microscopic phenotype analysis
Organisms Homo sapiens