Computational protocol: IglC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells

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Protocol publication

[…] A Leica DM4000B inverted fluorescence microscope attached to an Angstrom Grid Confocal system [Quorum Technologies] was used to view the samples and Metamorph Software was used to capture the images. ImageJ version 1.44i ( was used to count the number of invaded bacteria from images captured by either epifluorescence or structured illumination microscopy. Adobe Photoshop CS5 was used to process immunofluorescence images without changing the integrity of the data. To calculate the population of cells in each image, we semi-automatically measured the number of DAPI-stained cell nuclei using the “nucleus counter” plug-in, which is part of the McMaster Biophotonics Facility ImageJ bundle ( In the nucleus counter plug-in, the following parameters were adjusted to tally the number of nuclei in each image [taken at 40X magnification]: 2000, ‘Smallest Particle Size’; 6000, ‘Largest Particle Size’, Mean 3×3, ‘Smooth method’; enabled, ‘Watershed filter’. In case there were any miscounted nuclei or multi-nucleated cells, each image was further reviewed by overlaying the DAPI-stained image with the phase-contrast image. Within the population of adherent hepatocytes, infected and uninfected cells were manually tallied. Each infected cell was given a score of ‘1’ based on the presence of F. novicida within the cell boundaries, whereas each uninfected cell that had an absence of intracellular F. novicida was given a score of ‘0’. The proportion of infected cells was calculated based on the number of infected cells divided by the total number of cells in each image. To determine whether Francisella was within a LAMP1 containing vacuole, we looked for bacteria that were at least 50% surrounded or completely co-localized with LAMP1.Statistical analysis was performed using Graphpad Prism 6 software. Statistical significance was calculated using one-way ANOVA followed by Bonferroni multiple comparison test for data in Fig. 1, 2 and 3. Data in Fig. 4 and 7 were analyzed by Two-way ANOVA followed by Bonferroni multiple comparison test, whereas for Fisher’s LSD test was used for data in Fig. 8. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Francisella tularensis, Francisella tularensis subsp. novicida
Diseases Tularemia