Computational protocol: CLIQ-BID: A method to quantify bacteria-induced damage to eukaryotic cells by automated live-imaging of bright nuclei

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Protocol publication

[…] Cells were washed, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 5 min and labelled with a mouse anti-Vinculin 7F9 primary antibody (Santa Cruz) and donkey anti-mouse Cy3 secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h each. Nuclei were labelled with Hoechst 33258 (10 µg/ml, Sigma-Aldrich). Under these conditions, total cellular vinculin is detected yielding a whole-cell labelling. Images were captured using an automated microscope ArrayScanVTI with the 5x magnification objective, in the BGRFR-549/15 nm channel, and treated with ImageJ software. Briefly, images of vinculin staining were binarized and the total cell area was calculated for each image. To facilitate interpretation, results were shown as the percentage of the field area cleared by the cells. [...] To evaluate the quality of the assay and its ability to identify “hits”, the Z’-factor was calculated using the following equation, as described by Zhang et al.:1Z'=1−3∗(σp+σn)/|μp−μn|where σ p and σ n are the standard deviations of the positive and negative conditions, respectively, and μ p and μ n are the means of the positive and negative conditions, respectively.Statistical analyses were performed using SigmaPlot 12.5 (Systat software) for the comparison of multiple groups by one-way ANOVA (two-tailed). When appropriate, post hoc tests were Tukey or Dunnett for multiple comparisons or comparison to the control group, respectively.In figure legends, n represents the number of well replicates.Curve fitting was done with SigmaPlot 12.5 using the following equation:2y=y0+a1+e−b(x−x0)where y0 and a are the minimal and the range values of the bright nuclei percentage, respectively, and x0 and b respectively correspond to the inflection time and the curve steepness. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Bacteria
Diseases Bacterial Infections, Nervous System Diseases