Aug 06, 2020
Quantification of tube topography on micrographs of fluorescent staining of multi-layers cell culture.
- 1[Metcela Inc.]
External link: https://doi.org/10.1371/journal.pone.0237810
Protocol Citation: Bertrand-David Ségard 2020. Quantification of tube topography on micrographs of fluorescent staining of multi-layers cell culture.. protocols.io https://dx.doi.org/10.17504/protocols.io.bhtaj6ie
Manuscript citation:
Iwamiya T, Segard B, Matsuoka Y, Imamura T (2020) Human cardiac fibroblasts expressing VCAM1 improve heart function in postinfarct heart failure rat models by stimulating lymphangiogenesis. PLoS ONE 15(9): e0237810. doi: 10.1371/journal.pone.0237810
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our workspace and it is working.
Created: June 23, 2020
Last Modified: August 06, 2020
Protocol Integer ID: 38466
Keywords: co-culture, fluorescent staining, tube topography, quantification
Abstract
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).
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.
The present protocol allows to analyze the topography of tubes on micrographs of fluorescent staining of multi-layers cell culture.
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.
Software
Software
Install Icy (requires JDK 8+).
Software
Icy
NAME
Windows/MacOS/Linux
OS
BioImage Analysis Lab (Institut Pasteur)
DEVELOPER
SOURCE LINK
Download the Icy script "ImageJ background subtraction." (file name: protocolfile-imagej-background-subtraction1)
Install the Icy plugin "Membrane Filter."
Install the ImageJ plugin "Luts Macros and Tools Updater."
Install the ImageJ plugin "Angiogenesis Analyzer for ImageJ."
Preprocessing
Preprocessing
In Icy, open the image to be analyzed.
Extract the channel of interest (Channel -> Extract -> Channel x).
Save the new image as TIFF.
Subtract the background (Tools -> Protocols -> Load: protocolfile-imagej-background-subtraction1).
Settings: Rolling=20.
Save the new image as TIFF.
Reduce the discontinuities and noise on membranes (Plugin -> Membrane Filter).
Settings: default parameters.
Save the new image as TIFF.
Definition of ROI
Definition of ROI
In ImageJ (in Icy), open the image obtained after preprocessing.
Enhance the contrast (Process -> Enhance Contrast).
Settings: Saturated pixels: 0.0%; Normalize; Equalize histogram.
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
Analysis of tube topography
In ImageJ (in Icy), open the image obtained after the definition of ROI.
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.
Apply the Blurred Mask Tool (Angiogenesis Analyzer -> Blurred Mask Tool) to the whole surface of the image.
Change the color encoding of the image to RGB color (Image -> Type -> RGB color).
Run the analysis (Angiogenesis Analyzer -> Analyze HUVEC Phase contrast).
Save and analyze
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.