TITLE: Quantification of tube topography on micrographs of fluorescent staining of multi-layers cell culture.
AUTHORS: Bertrand-David Segard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">The induction or inhibition of angiogenesis can be assessed in vitro by the differentiation of endothelial cells. Indeed, these cells are capable of forming structures resembling a capillary network in culture. This approach is known as Endothelial Tube Formation Assay (ETFA).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">This type of analysis is traditionally performed on a low confluence monolayer of cells to assay the effect of soluble compounds on the formation of tubes. However, the modulation of tube formation by cell-cell interactions requires the co-culture of cells that leads to the creation of a multi-layer micro-tissue. Additionally, the identification of cells in the micro-tissue requires fluorescent staining resulting in discontinuities in the signal.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The present protocol allows to analyze the topography of tubes on micrographs of fluorescent staining of multi-layers cell culture.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The limit of this method resides in the fact that the 3D organization of the micro-tissue is not reconstructed and, thus, a part of the tube network is not analyzed.</div></div></div>

[STEPS]
?. [Software]
Install Icy (requires JDK 8+).
?. [Software]
Download the Icy script "ImageJ background subtraction." (file name: protocolfile-imagej-background-subtraction1)See: http://icy.bioimageanalysis.org/protocol/imagej-background-subtraction/
?. [Software]
Install the Icy plugin "Membrane Filter."See: http://icy.bioimageanalysis.org/plugin/membrane-filter/
?. [Software]
Install the ImageJ plugin "Luts Macros and Tools Updater."See: http://image.bio.methods.free.fr/ImageJ/?Luts-Macros-and-Tools-Updater
?. [Software]
Install the ImageJ plugin "Angiogenesis Analyzer for ImageJ."See: http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ
?. [Preprocessing]
In Icy, open the image to be analyzed.
?. [Preprocessing]
Extract the channel of interest (Channel -> Extract -> Channel x).Save the new image as TIFF.
?. [Preprocessing]
Subtract the background (Tools -> Protocols -> Load: protocolfile-imagej-background-subtraction1).Settings: Rolling=20.Save the new image as TIFF.
?. [Definition of ROI]
In ImageJ (in Icy), open the image obtained after preprocessing.
?. [Definition of ROI]
Enhance the contrast (Process -> Enhance Contrast).Settings: Saturated pixels: 0.0%; Normalize; Equalize histogram.
?. [Definition of ROI]
Subtract the background (Process -> Subtract Background).Settings: Rolling ball radius: 5.0 pixels; Sliding paraboloid.Save the new image as TIFF.
?. [Analysis of tube topography]
In ImageJ (in Icy), open the image obtained after the definition of ROI.
?. [Analysis of tube topography]
Start Angiogenesis Analyzer.Settings: select all options except "Suppress isolated elements."Threshold values: Minimum object size: 10 pixels; Minimum branch size: 25 pixels; Artifactual loop size: 850 pixels; Isolated element size threshold: 50 pixels; Master segment size threshold: 30 pixels; Iteration number (advised 2 to 5): 3 iterations; Show iteration (for single analysis): 3 iterations.
?. [Analysis of tube topography]
Apply the Blurred Mask Tool (Angiogenesis Analyzer -> Blurred Mask Tool) to the whole surface of the image.
?. [Analysis of tube topography]
Change the color encoding of the image to RGB color (Image -> Type -> RGB color).
?. [Analysis of tube topography]
Run the analysis (Angiogenesis Analyzer -> Analyze HUVEC Phase contrast).
?. [Save and analyze]
Save the result files (Angiogenesis Analyzer -> Save Current Analysis). Measurements are saved as a tab-separated values file (with "xls" extension) and can be further analyzed in a spreadsheet.
?. [Preprocessing]
Reduce the discontinuities and noise on membranes (Plugin -> Membrane Filter).Settings: default parameters.Save the new image as TIFF.