After establishing the stromal endothelial cell co-culture, acquire the GFP signal from the GFP huvec using fluorescence microscopy with settings suitable for quantification. Pre-process all the images acquired on the same day to further enhance the contrast. If using fiji or image J open all the enhanced GFP channel images at the same time point and open the brightness and contrast menu.
Select an image representing an intermediate condition and auto adjust the contrast by selecting auto. Click set and check propagate to all other open images. Visually assess whether the automatically selected range fits all the images of the current time point.
If needed, manually readjust and repropagate the range to all the images and save the adjusted images as TIFF files. Next, apply a median blur filter to all the images. Reduce the size by binning and save them as gray scale RGB color TIFF files in a folder for quantification.
This could be done manually or in batch mode using macros. Analyze all the created images using the batch process mode in the angiogenesis analyzer for image J.Then validate the quantification results by examining the overlays of the recognized structures and the original images. Adjust pre-processing parameters, reanalyze original images, or exclude problematic areas if the algorithm detects artificial structures with few or no cells visible in the original image.
Finally normalize the obtained values to an area of one square millimeter by multiplying the values of each sample by the ratio of the analyzed area to one square millimeter. Quantified parameters of the GFP huvec networks showed that the total network length was highest in the presence of FGF-2 and lowest in the absence of growth factors. The number of junctions indicating branching points in the networks followed the same trend as the total length.
Conversely, both growth factors featured significantly fewer isolated segments indicating higher interconnectivity than the condition without any growth factor.
